Ube2t peptides and vaccines containing the same

ABSTRACT

Peptide vaccines against cancer are described herein. In particular, epitope peptides derived from the UBE2T that CTLs are provided. Isolated antigen-presenting cells with CTL inducibility and CTLs that target such peptides, as well as methods for inducing the antigen-presenting cell, or CTL are also provided. The present invention further provides pharmaceutical compositions containing such epitope peptides derived from UBE2T or polynucleotides encoding the polypeptides as active ingredients. Furthermore, the present invention provides methods for the treatment and/or prophylaxis of (i.e., preventing) cancers (tumors), and/or the prevention of a postoperative recurrence thereof, as well as methods for inducing CTLs, methods for inducing anti-tumor immunity, using the epitope peptides derived from UBE2T, polynucleotides encoding the peptides, or antigen-presenting cells presenting the peptides, or the pharmaceutical compositions of the present invention.

PRIORITY

The present application claims the benefit of U.S. ProvisionalApplication No. 61/699,550, filed on Sep. 11, 2012, the entire contentsof which are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer therapy. In particular, the presentinvention relates to novel peptides that are effective as cancervaccines, as well as drugs for either or both of treating and preventingtumors.

BACKGROUND ART

It has been demonstrated that CD8 positive cytotoxic T lymphocytes(CTLs) recognize epitope peptides derived from the tumor-associatedantigens (TAAs) found on the major histocompatibility complex (MHC)class I molecule, and then kill the tumor cells. Since the discovery ofthe melanoma antigen (MAGE) family as the first example of TAAs, manyother TAAs have been discovered, primarily through immunologicalapproaches (NPL 1-2). Some of these TAAs are currently undergoingclinical development as immunotherapeutic targets.

Favorable TAAs are indispensable for the proliferation and survival ofcancer cells. The use of such TAAs as targets for immunotherapy mayminimize the well-described risk of immune escape of cancer cellsattributable to deletion, mutation, or down-regulation of TAAs as aconsequence of therapeutically driven immune selection. Accordingly, theidentification of new TAAs capable of inducing potent and specificanti-tumor immune responses warrants further development and thusclinical application of peptide vaccination strategies for various typesof cancer in ongoing (NPL 3-10). To date, there have been severalreports of clinical trials using these TAAs-derived peptides.Unfortunately, only a low objective response rate has been observed inthese cancer vaccine trials (NPL 11-13). Accordingly, there remains aneed for new TAAs as immunotherapeutic targets.

UBE2T (ubiquitin-conjugating enzyme E2T: a typical amino acid sequenceshown in SEQ ID NO: 65; a typical nucleotide sequence shown in SEQ IDNO: 64 (GenBank Accession No. NM_014176)) is one of theubiquitin-conjugating enzymes (E2). UBE2T was reported to be one of thegenes whose expression was up-regulated in human fibroblasts with serumstimulation (NPL 14). In the Fanconi anemia pathway, UBE2T binds FANCL,and is necessary for the DNA damage-induced monoubiquitination of FANCD2(NPL 15-16). In recent studies, UBE2T was found to be frequentlyup-regulated in breast cancers, and interact and co-localize with theBRCA1/BRCA1-associated RING domain protein (BARD1) complex (PTL 1, NPL17). Northern blot analysis in those studies revealed that UBE2Ttranscript was detected at very high level in breast cancer cell lines,but hardly detected in the vital organs. Furthermore, knockdowns ofendogenous UBE2T by siRNA in cancer cell lines have been shown tosignificantly suppress growth of those cell lines (PTL 1-2, NPL 17).

CITATION LIST Patent Literature

[PTL 1] WO2005/029067

[PTL 2] WO2009/001562

Non Patent Literature

[NPL 1] Boon T, Int J Cancer 1993, 54(2): 177-80

[NPL 2] Boon T & van der Bruggen P, J Exp Med 1996, 183(3): 725-9

[NPL 3] Harris C C, J Natl Cancer Inst 1996, 88(20): 1442-55

[NPL 4] Butterfield L H et al., Cancer Res 1999, 59(13): 3134-42

[NPL 5] Vissers J L et al., Cancer Res 1999, 59(21): 5554-9

[NPL 6] van der Burg S H et al., J Immunol 1996, 156(9): 3308-14

[NPL 7] Tanaka F et al., Cancer Res 1997, 57(20): 4465-8

[NPL 8] Fujie T et al., Int J Cancer 1999, 80(2): 169-72

[NPL 9] Kikuchi M et al., Int J Cancer 1999, 81(3): 459-66

[NPL 10] Oiso Metal., Int J Cancer 1999, 81(3): 387-94

[NPL 11] Belli F et al., J Clin Oncol 2002, 20(20): 4169-80

[NPL 12] Coulie P G et al., Immunol Rev 2002, 188: 33-42

[NPL 13] Rosenberg S A et al., Nat Med 2004, 10(9): 909-15

[NPL 14] Iyer V R et al., Science 1999, 283: 83-7

[NPL 15] Machida Y J et al., Mol Cell 2006, 23: 589-96

[NPL 16] Alpi A et al., Mol Cell Biol 2007, 27: 8421-30

[NPL 17] Ueki T et.al., Cancer Res. 2009, 69: 8752-60

SUMMARY OF INVENTION

The present invention is based, at least in part, on the discovery ofnovel peptides that may serve as suitable targets of immunotherapy.Because TAAs are generally perceived by the immune system as “self” andtherefore often have no innate immunogenicity, the discovery ofappropriate targets is still of importance.

To that end, the present invention is directed, at least in part, to theidentification of specific epitope peptides that possess the ability toinduce CTLs specific to UBE2T among peptides derived from UBE2T.

The results disclosed herein demonstrate that identified peptides areHLA-A24 or HLA-A2 restricted epitope peptides that can induce potent andspecific immune responses against cells expressing UBE2T.

Accordingly, it is an object of the present invention to provideUBE2T-derived peptides that can be used to induce CTLs in vitro, ex vivoor in vivo, or to be administered to a subject for inducing immuneresponses against cancers, examples of which include, but are notlimited to, bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, esophageal cancer,gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor. Preferred peptides arenonapeptides and decapeptides, more preferably nonapeptides anddecapeptides having an amino acid sequence selected from among SEQ IDNOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29,30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58. Of these, thepeptides having an amino acid sequence selected from among SEQ ID NOs:1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30,32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58 are particularlypreferred.

The present invention also contemplates modified peptides having anamino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 6, 11, 12,13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38, 41, 48,49, 51, 52, 53, 55, 56 and 58 in which one, two or more amino acids aresubstituted, deleted, inserted and/or added, as long as the resultingmodified peptides retain the requisite CTL inducibility of the originalunmodified peptide. In one embodiment, when the original peptides is9mer (SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 29, 30, 32, 36, 38, and41), the size of the modified peptide is preferably in the range of 9 to40 amino acids, such as in the range of 9 to 20 amino acids, for examplein the range 9 to 15 amino acids. Likewise, when the original peptidesis 10mer (SEQ ID NOs: 17, 19, 20, 21, 22, 23, 24, 25, 27, 48, 49, 51,52, 53, 55, 56 and 58), the size of the modified peptide is preferablyin the range of 10 to 40 amino acids, such as in the range of 10 to 20amino acids, for example in the range 10 to 15 amino acids.

The present invention further encompasses isolated polynucleotidesencoding any one of the peptides of the present invention. Thesepolynucleotides can be used to induce or prepare antigen-presentingcells (APCs)having CTL inducibility. Like the peptides of the presentinvention, such APCs can be administered to a subject for inducingimmune responses against cancers.

When administered to a subject, the peptides of the present inventioncan be presented on the surface of APCs so as to induce CTLs targetingthe respective peptides. Therefore, one object of the present inventionis to provide agents or compositions including one or more peptides ofthe present invention, or polynucleotides encoding such peptides. Theagent or composition may be used for inducing a CTL. Such agents orcompositions can be used for the treatment and/or prophylaxis of acancer, and/or the prevention of a metastasis or post-operativerecurrence thereof. Examples of cancers contemplated by the presentinvention include, but are not limited to, bladder cancer, breastcancer, cervical cancer, cholangiocellular carcinoma, CML, colorectalcancer, esophageal cancer, gastric cancer, diffuse-type gastric cancer,NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer,prostate cancer, SCLC, soft tissue tumor and testicular tumor.

The present invention further contemplates pharmaceutical compositionsor agents that include one or more peptides or polynucleotides of thepresent invention. The pharmaceutical composition is preferablyformulated for use in the treatment and/or prophylaxis of a cancer,and/or prevention of metastasis or postoperative recurrence thereof.Instead of or in addition to the peptides or polynucleotides of thepresent invention, the pharmaceutical agents or compositions of thepresent invention may include as active ingredients APCs and/or exosomesthat present any of the peptides of the present invention.

The peptides or polynucleotides of the present invention may be used toinduce APCs that present on the surface a complex of a human leukocyteantigen (HLA) and a peptide of the present invention, for example, bycontacting APCs derived from a subject with the peptide of the presentinvention or introducing a polynucleotide encoding the peptide of thepresent invention into APCs. Such APCs have the ability to induce CTLsthat specifically recognize cells that present target peptides on thesurface and are useful for cancer immunotherapy. Accordingly, thepresent invention encompasses the methods for inducing APCs with CTLinducibility as well as the APCs obtained by such methods.

In addition, the present invention also encompasses the agents orcompositions for inducing APCs having CTL inducibility, such agents orcompositions including any peptides or polynucleotides of the presentinvention.

It is a further object of the present invention to provide methods forinducing CTLs, such methods including the step of co-culturing CD8positive T cells with APCs presenting on its surface a complex of an HLAantigen and the peptide of the present invention, the step ofco-culturing CD8 positive T cells with exosomes presenting on itssurface a complex of an HLA antigen and the peptide of the presentinvention, or the step of introducing a polynucleotide encoding both ofT cell receptor (TCR) subunits or polynucleotides encoding each of TCRsubunits, wherein the TCR can bind to a complex of the peptide of thepresent invention and an HLA antigen presented on cell surface. CTLsobtained by such methods can find use in the treatment and/or preventionof cancers, examples of which include, but are not limited to, bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, esophageal cancer, gastric cancer, diffuse-typegastric cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer,pancreatic cancer, prostate cancer, SCLC, soft tissue tumor andtesticular tumor. Therefore, the present invention also encompasses CTLsobtained by above-described methods.

Yet another object of the present invention is to provide isolated APCsthat present on the surface a complex of an HLA antigen and a peptide ofthe present invention. The present invention further provides isolatedCTLs that target peptides of the present invention. Such CTLs may bealso defined as CTLs that can recognize (or bind to) a complex of apeptide of the present invention and an HLA antigen on the cell surface.These APCs and CTLs may be used for cancer immunotherapy.

It is yet another object of the present invention to provide methods forinducing an immune response against a cancer in a subject in needthereof, such methods including the step of administering to the subjectan agent or composition including at least one component selected fromamong a peptide of the present invention or a polynucleotide encodingthereof, an APC or exosome presenting thereof and a CTL that canrecognize a cell presenting the peptide of the present invention on thesurface.

One aspect of the present invention pertains to a peptide of the presentinvention or a composition comprising a peptide of the present inventionfor use a medicament.

The applicability of the present invention extends to any of a number ofdiseases relating to or arising from UBE2T overexpression, such ascancer, examples of which include, but are not limited to, bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, esophageal cancer, gastric cancer, diffuse-typegastric cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer,pancreatic cancer, prostate cancer, SCLC, soft tissue tumor andtesticular tumor.

More specifically, the present invention provides followings:

[1] An isolated peptide having cytotoxic T lymphocyte (CTL)inducibility, wherein the peptide comprises an amino acid sequence (a)or (b) bellow:

(a) an amino acid sequence of an immunologically active fragment ofUBE2T;

(b) an amino acid sequence in which 1, 2, or several amino acid(s) aresubstituted, deleted, inserted and/or added in an amino acid sequence ofan immunologically active fragment of UBE2T,

wherein the CTL induced by the peptide has specific cytotoxic activityagainst a cell that presents a fragment derived from UBE2T;

[2] The peptide of [1], wherein the peptide comprises an amino acidsequence (a) or

(b) bellow:

(a) an amino acid sequence selected from the group consisting of SEQ IDNOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29,30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58;

(b) an amino acid sequence in which 1, 2, or several amino acid(s) aresubstituted, deleted, inserted and/or added in the amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 4, 6, 11, 12,13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38, 41, 48,49, 51, 52, 53, 55, 56 and 58; The size of the modified peptide ispreferably in the range of 9 to 40 amino acids, such as in the range of9 to 20 amino acids, for example in the range 9 to 15 amino acids;

[3] The peptide of [2], wherein the peptide is the followingoligopeptide (i) or (ii):

(i) a peptide that has one or both of the following characteristics:

(a) the second amino acid from the N-terminus of the amino acid sequenceof SEQ ID NO: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25or 27 is substituted with phenylalanine, tyrosine, methionine ortryptophan; and

(b) the C-terminal amino acid of the amino acid sequence of SEQ ID NO:1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25 or 27 issubstituted with phenylalanine, leucine, isoleucine, tryptophan ormethionine;

(ii) a peptide that has one or both of the following characteristics:

(a) the second amino acid from the N-terminus of the amino acid sequenceof SEQ ID NO: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58is substituted with leucine or methionine; and

(b) the C-terminal amino acid of the amino acid sequence of SEQ ID NO:29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58 is substitutedwith valine or leucine;

[4] The peptide of any one of [1] to [3], wherein the peptide is anonapeptide or a decapeptide;

[5] The peptide of [4], wherein the peptide consists of the amino acidsequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 6,11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38,41, 48, 49, 51, 52, 53, 55, 56 and 58;

[6] An isolated polynucleotide encoding the peptide of any one of [1] to[5];

[7] A composition for inducing a CTL, wherein the composition comprisesat least one active ingredient selected from the group consisting of:

(a) the peptide of any one of [l ] to [5];

(b) the polynucleotide of [6];

(c) an APC that presents the peptide of any one of [1] to [5] on itssurface; and

(d) an exosome that presents the peptide of any one of [1] to [5] on itssurface;

[8] A pharmaceutical composition for the treatment and/or prophylaxis ofcancer, and/or the prevention of a postoperative recurrence thereof,wherein the composition comprises at least one active ingredientselected from the group consisting of:

(a) the peptide of any one of [1] to [5];

(b) the polynucleotide of [6];

(c) an APC that presents the peptide of any one of [1] to [5] on itssurface

(d) an exosome that presents the peptide of any one of [1] to [5] on itssurface; and

(e) a CTL that can recognize a cell presenting the peptide of any one of[1] to [5];

[9] The pharmaceutical composition of [8], wherein the pharmaceuticalcomposition is formulated for the administration to a subject whose HLAantigen is HLA-A24 or HLA-A2;

[10] A method for inducing an APC with CTL inducibility, wherein themethod comprises the step selected from the group consisting of:

(a) contacting an APC with the peptide of any one of [1] to [5], and

(b) introducing a polynucleotide encoding the peptide of any one of [1]to [5] into an APC;

[11] A method for inducing a CTL, wherein the method comprises a stepselected from the group consisting of:

(a) co-culturing a CD8 positive T cell with an APC that presents on itssurface a complex of an HLA antigen and the peptide of any one of [1] to[5];

(b) co-culturing a CD8 positive T cell with an exosome that presents onits surface a complex of an HLA antigen and the peptide of any one of[1] to [5]; and

(c) introducing into a CD8 positive T cell a polynucleotide encodingboth of TCR subunits or polynucleotides encoding each of TCR subunits,wherein the TCR formed by said subunits can bind to a complex of thepeptide of any one of [1] to [5] and an HLA antigen on a cell surface;

[12] An isolated APC that presents on its surface a complex of an HLAantigen and the peptide of any one of [1] to [5];

[13] The APC of [12], which is induced by the method of [10];

[14] An isolated CTL that targets the peptide of any one of [1] to [5];

[15] The CTL of [14], which is induced by the method of [11];

[16] A method of inducing an immune response against cancer in asubject, wherein the method comprises the step of administering to thesubject a composition comprising the peptide of any one of [1] to [5],or a polynucleotide encoding the peptide;

[17] An antibody or immunologically active fragment thereof against thepeptide of any one of [1] to [5];

[18] A vector comprising a nucleotide sequence encoding the peptide ofany one of [1] to [5]; Preferably the vector is adapted for expressionof said peptide (referred to as expression vector), e.g. the encodingnucleotide sequence is inserted in vector downstream of a promotersequence and operably linked to said promoter sequence. The term“operably linked” is intended to mean that the nucleotide sequence islinked to the promoter sequence (regulatory sequence) such that itallows expression of the nucleotide sequence in vitro or in a host cellin which the vector is introduced;

[19] A host cell transformed or transfected with a vector of [18] orexpression vector described herein;

[20] A diagnostic kit comprising the peptide of any one of [1] to [5],the polynucleotide of [6] or the antibody or immunologically activefragment of [17]; and

[21] A method of screening for a peptide having an ability to induce aCTL that has specific cytotoxic activity against a cell that presents afragment derived from UBE2T, wherein the method comprises the steps of:

(i) providing a candidate sequence consisting of an amino acid sequencemodified by substituting, deleting, inserting and/or adding one, two orseveral amino acid residues to an original amino acid sequence, whereinthe original amino acid sequence is selected from the group consistingof SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24,25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58;

(ii) selecting a candidate sequence that does not have substantialsignificant homology (or sequence identity) with the peptides derivedfrom any known human gene products other than UBE2T;

(iii) contacting a peptide consisting of the candidate sequence selectedin step (ii) with an antigen presenting cell;

(iv) contacting the antigen presenting cell of step (iii) with a CD8positive T cell; and

(v) identifying the peptide of which CTL inducibility is same to orhigher than a peptide consisting of the original amino acid sequence.

[22] A pharmaceutical composition comprising peptide of any one of [1]to [5].

[23] A peptide of any one of [1] to [5] for use as a medicament.

[24] A polynucleotide of [6] or a vector of [18] for use as amedicament.

In addition to the above, other objects and features of the inventionwill become more fully apparent when the following detailed descriptionis read in conjunction with the accompanying figures and examples.However, it is to be understood that both the foregoing summary of theinvention and the following detailed description are of exemplifiedembodiments, and not restrictive of the invention or other alternateembodiments of the invention.

In particular, while the invention is described herein with reference toa number of specific embodiments, it will be appreciated that thedescription is illustrative of the invention and is not constructed aslimiting of the invention. Various modifications and applications mayoccur to those who are skilled in the art, without departing from thespirit and the scope of the invention, as described by the appendedclaims. Likewise, other objects, features, benefits and advantages ofthe present invention will be apparent from this summary and certainembodiments described below, and will be readily apparent to thoseskilled in the art. Such objects, features, benefits and advantages willbe apparent from the above in conjunction with the accompanyingexamples, data, figures and all reasonable inferences to be drawntherefrom, alone or with consideration of the references incorporatedherein.

BRIEF DESCRIPTION OF DRAWINGS

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the presentinvention and its preferred embodiments that follows.

FIG. 1a -1 is composed of a series of photographs, (a) to (l), showingthe results of interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT)assay on CTLs that were induced with peptides derived from UBE2T. TheCTLs in the well number #8 stimulated with UBE2T-A24-9-60 (SEQ ID NO: 1)(a), #1 stimulated with UBE2T-A24-9-45 (SEQ ID NO: 2) (b), #6 stimulatedwith UBE2T-A24-9-133 (SEQ ID NO: 4) (c), #6 stimulated withUBE2T-A24-9-138 (SEQ ID NO: 6) (d), #4 stimulated with UBE2T-A24-9-43(SEQ ID NO: 11) (e), #2 stimulated with UBE2T-A24-9-106 (SEQ ID NO: 12)(f), #6 stimulated with UBE2T-A24-9-3 (SEQ ID NO: 13) (g), #3 stimulatedwith UBE2T-A24-9-105 (SEQ ID NO: 15) (h), #2 stimulated withUBE2T-A24-10-130 (SEQ ID NO: 17) (i), #1 stimulated withUBE2T-A24-10-131 (SEQ ID NO: 19) (j), #3 stimulated withUBE2T-A24-10-133 (SEQ ID NO: 20) (k), and #6 stimulated withUBE2T-A24-10-99 (SEQ ID NO: 21) (l) showed potent IFN-gamma productioncompared with the control, respectively. The square on the well of thesepictures indicates that the cells from corresponding well were expandedto establish CTL lines. In contrast, as a typical case of a negativedata, specific IFN-gamma production from the CTL stimulated withUBE2T-A24-9-124 (SEQ ID NO: 3) (r) was not shown. In the figures, “+”indicates the IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides.

FIG. 1m-r is composed of a series of photographs, (m) to (r), showingthe results of interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT)assay on CTLs that were induced with peptides derived from UBE2T. TheCTLs in the well number #7 stimulated with UBE2T-A24-10-154 (SEQ ID NO:22) (m), #8 stimulated with UBE2T-A24-10-105 (SEQ ID NO: 23) (n), #1stimulated with UBE2T-A24-10-115 (SEQ ID NO: 24) (o), #4 stimulated withUBE2T-A24-10-177 (SEQ ID NO: 25) (p) and #7 stimulated withUBE2T-A24-10-44 (SEQ ID NO: 27) (q) showed potent IFN-gamma productioncompared with the control, respectively. The square on the well of thesepictures indicates that the cells from corresponding well were expandedto establish CTL lines. In contrast, as a typical case of a negativedata, specific IFN-gamma production from the CTL stimulated withUBE2T-A24-9-124 (SEQ ID NO: 3) (r) was not shown. In the figures, “+”indicates the IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides.

FIG. 2a-l is composed of a series of photographs, (a) to (l), showingthe results of ELISPOT assay on CTLs that were induced with peptidesderived from UBE2T. The CTLs in the well number #4 stimulated withUBE2T-A02-9-107 (SEQ ID NO: 29) (a), #5 stimulated with UBE2T-A02-9-30(SEQ ID NO: 30) (b), #7 stimulated with UBE2T-A02-9-106 (SEQ ID NO: 32)(c), #5 stimulated with UBE2T-A02-9-49 (SEQ ID NO: 36) (d), #3stimulated with UBE2T-A02-9-13 (SEQ ID NO: 38) (e), #4 stimulated withUBE2T-A02-9-132 (SEQ ID NO: 41) (f), #6 stimulated with UBE2T-A02-10-70(SEQ ID NO: 48) (g), #7 stimulated with UBE2T-A02-10-6 (SEQ ID NO: 49)(h), #8 stimulated with UBE2T-A02-10-106 (SEQ ID NO: 51) (i), #2stimulated with UBE2T-A02-10-102 (SEQ ID NO: 52) (j), #1 stimulated withUBE2T-A02-10-30 (SEQ ID NO: 53) (k), and #8 stimulated withUBE2T-A02-10-101 (SEQ ID NO: 55) (1) showed potent IFN-gamma productioncompared with the control, respectively. The square on the well of thesepictures indicates that the cells from corresponding well were expandedto establish CTL lines. In contrast, as a typical case of a negativedata, specific IFN-gamma production from the CTL stimulated withUBE2T-A02-9-161 (SEQ ID NO: 28) (o) was not shown. In the figures, “+”indicates the IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides.

FIG. 2m-o is composed of a series of photographs, (m) to (o), showingthe results of ELISPOT assay on CTLs that were induced with peptidesderived from UBE2T. The CTLs in the well number #5 stimulated withUBE2T-A02-10-29 (SEQ ID NO: 56) (m) and #3 stimulated withUBE2T-A02-10-38 (SEQ ID NO: 58) (n) showed potent IFN-gamma productioncompared with the control, respectively. The square on the well of thesepictures indicates that the cells from corresponding well were expandedto establish CTL lines. In contrast, as a typical case of a negativedata, specific IFN-gamma production from the CTL stimulated withUBE2T-A02-9-161 (SEQ ID NO: 28) (o) was not shown. In the figures, “+”indicates the IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides.

FIG. 3 is composed of a series of line graphs, (a) to (d), showing theIFN-gamma production of the CTL lines stimulated with UBE2T-A24-9-60(SEQ ID NO: 1) (a), UBE2T-A24-9-45 (SEQ ID NO: 2) (b), UBE2T-A24-9-3(SEQ ID NO: 13) (c) and UBE2T-A24-10-44 (SEQ ID NO: 27) (d). Thequantity of IFN-gamma which CTLs produced was measured by IFN-gammaenzyme-linked immunosorbent assay (ELISA). The results demonstrates thatCTL lines established by stimulation with each peptide show potentIFN-gamma production as compared with the control. In the figures, “+”indicates the IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides. R/S ratio indicates the ratioof the number of responder cells (CTL line) and stimulator cells.

FIG. 4 is composed of a series of line graphs, (a) to (c), showing theIFN-gamma production of the CTL clones established by limiting dilutionfrom the CTL lines stimulated with UBE2T-A24-9-60 (SEQ ID NO: 1) (a),UBE2T-A24-9-45 (SEQ ID NO: 2) (b) and UBE2T-A24-9-3 (SEQ ID NO: 13) (c).The results demonstrate that the CTL clones established by stimulationwith each peptide show potent IFN-gamma production as compared with thecontrol. In the figures, “+” indicates the IFN-gamma production againsttarget cells pulsed with the appropriate peptide and “−” indicates theIFN-gamma production against target cells not pulsed with any peptides.R/S ratio indicates the ratio of the number of responder cells (CTLclone) and stimulator cells.

FIG. 5 is composed of a series of line graphs, (a) to (e), showing theIFN-gamma production of the CTL lines stimulated with UBE2T-A02-9-107(SEQ ID NO: 29) (a), UBE2T-A02-9-13 (SEQ ID NO: 38) (b), UBE2T-A02-10-70(SEQ ID NO: 48) (c), UBE2T-A02-10-102 (SEQ ID NO: 52) (d) andUBE2T-A02-10-101 (SEQ ID NO: 55) (e). The quantity of IFN-gamma whichCTL produced was measured by IFN-gamma ELISA. The results demonstratethat CTL lines established by stimulation with each peptide show potentIFN-gamma production as compared with the control. In the figures, “+”indicates the IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides. R/S ratio indicates the ratioof the number of responder cells (CTL line) and stimulator cells.

FIG. 6 is composed of series of line graphs, (a) to (e), showing theIFN-gamma production of the CTL clones established by limiting dilutionfrom the CTL lines stimulated with UBE2T-A02-9-107 (SEQ ID NO: 29) (a),UBE2T-A02-9-13 (SEQ ID NO: 38) (b), UBE2T-A02-10-70 (SEQ ID NO: 48) (c),UBE2T-A02-10-102 (SEQ ID NO: 52) (d) and UBE2T-A02-10-101 (SEQ ID NO:55) (e). The results demonstrate that the CTL clones established bystimulation with each peptide show potent IFN-gamma production ascompared with the control. In the figures, “+” indicates the IFN-gammaproduction against target cells pulsed with the appropriate peptide and“−” indicates the IFN-gamma production against target cells not pulsedwith any peptides. R/S ratio indicates the ratio of the number ofresponder cells (CTL clone) and stimulator cells.

FIG. 7 is a line graph, showing specific CTL activity against the targetcells that express UBE2T and HLA-A*2402. COS7 cells transfected withHLA-A*2402 or the full length of the UBE2T gene were prepared ascontrols. The CTL clone established with UBE2T-A24-9-60 (SEQ ID NO: 1)showed specific CTL activity against COS7 cells transfected with bothUBE2T and HLA-A*2402 (black lozenge). On the other hand, no significantspecific CTL activity was detected against target cells expressingeither HLA-A*2402 (triangle) or UBE2T (circle).

FIG. 8 is a line graph, showing specific CTL activity against the targetcells that express UBE2T and HLA-A*0201. COS7 cells transfected withHLA-A*0201 or the full length of the UBE2T gene were prepared ascontrols. The CTL line established with UBE2T-A02-10-70 (SEQ ID NO: 48)showed specific CTL activity against COS7 cells transfected with bothUBE2T and HLA-A*0201 (black lozenge). On the other hand, no significantspecific CTL activity was detected against target cells expressingeither HLA-A*0201 (triangle) or UBE2T (circle).

DESCRIPTION OF EMBODIMENTS

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. However, before the present materials and methods aredescribed, it should be understood that these descriptions are merelyillustrative and not intended to be limited. It should also beunderstood that the present invention is not limited to the particularsizes, shapes, dimensions, materials, methodologies, protocols, etc.described herein, as these may vary in accordance with routineexperimentation and optimization. Furthermore, the terminology used inthe description is for the purpose of describing the particular versionsor embodiments only, and is not intended to limit the scope of thepresent invention which will be limited only by the appended claims.

The disclosure of each publication, patent or patent applicationmentioned in this specification is specifically incorporated byreference herein in its entirety. However, nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue or prior invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present invention belongs. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

I. Definitions

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

The terms “isolated” and “purified” used in relation with a substance(e.g., peptide, antibody, polynucleotide, etc.) indicates that thesubstance is substantially free from at least one substance that mayelse be included in the natural source. Thus, an isolated or purifiedpeptide refers to a peptide that are substantially free of cellularmaterial such as carbohydrate, lipid, or other contaminating proteinsfrom the cell or tissue source from which the peptide is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The term “substantially free of cellularmaterial” includes preparations of a peptide in which the peptide isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a peptide that issubstantially free of cellular material includes preparations ofpolypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous protein (also referred to herein as a “contaminatingprotein”). When the peptide is recombinantly produced, it is alsopreferably substantially free of culture medium, which includespreparations of peptide with culture medium less than about 20%, 10%, or5% of the volume of the peptide preparation. When the peptide isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, which includes preparations ofpeptide with chemical precursors or other chemicals involved in thesynthesis of the peptide less than about 30%, 20%, 10%, 5% (by dryweight) of the volume of the peptide preparation. That a particularpeptide preparation contains an isolated or purified peptide can beshown, for example, by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamidegel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining or the like of thegel. In a preferred embodiment, peptides and polynucleotides of thepresent invention are isolated or purified.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue(s) may be modified residue(s), or non-naturally occurringresidue(s), such as artificial chemical mimetic(s) of correspondingnaturally occurring amino acid(s), as well as to naturally occurringamino acid polymers.

The term “oligopeptide” as used herein refers to a peptide which iscomposed of 20 amino acid residues or fewer, typically 15 amino acidresidues or fewer. As used herein, the term “nonapeptide” refers to apeptide which is composed of 9 amino acid residues and the term“decapeptide” refers to a peptide which is composed of 10 amino acidresides.

The term “amino acid” as used herein refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that similarly function to the naturally occurring amino acids.Amino acids may be either L-amino acids or D-amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose modified after translation in cells (e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine). The phrase “amino acidanalog” refers to compounds that have the same basic chemical structure(an alpha carbon bound to a hydrogen, a carboxy group, an amino group,and an R group) as a naturally occurring amino acid but have a modifiedR group or modified backbone (e.g., homoserine, norleucine, methionine,sulfoxide, methionine methyl sulfonium). The phrase “amino acid mimetic”refers to chemical compounds that have different structures but similarfunctions to general amino acids.

Amino acids may be referred to herein by their commonly known threeletter symbols or the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

The terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” areused interchangeably herein and, unless otherwise specificallyindicated, are referred to by their commonly accepted single-lettercodes.

The term “agent” and “composition” are used interchangeably herein torefer to a product including the specified ingredients in the specifiedamounts, as well as any product that results, directly or indirectly,from combination of the specified ingredients in the specified amounts.Such terms, when used in relation to the modifier “pharmaceutical” (asin “pharmaceutical agent” and “pharmaceutical composition”) are intendedto encompass a product that includes the active ingredient(s), and theinert ingredient(s) that make up the carrier, as well as any productthat results, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, in thecontext of the present invention, the terms “pharmaceutical agent” and“pharmaceutical composition” refer to any product made by admixing amolecule or compound of the present invention and a pharmaceutically orphysiologically acceptable carrier.

The term “active ingredient” herein refers to a substance in an agent orcomposition that is biologically or physiologically active.Particularly, in the context of pharmaceutical agent or composition, theterm “active ingredient” refers to a substance that shows an objectivepharmacological effect. For example, in case of pharmaceutical agents orcompositions for use in the treatment or prevention of cancer, activeingredients in the agents or compositions may lead to at least onebiological or physiological action on cancer cells and/or tissuesdirectly or indirectly. Preferably, such action may include reducing orinhibiting cancer cell growth, damaging or killing cancer cells and/ortissues, and so on. Typically, indirect effects of active ingredientsare inductions of CTLs that can recognize or kill cancer cells. Beforebeing formulated, the “active ingredient” may also be referred to as“bulk”, “drug substance” or “technical product”.

The phrase “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier”, as used herein, means a pharmaceutically orphysiologically acceptable material, composition, substance or vehicle,including, but are not limited to, a liquid or solid filler, diluent,excipient, solvent and encapsulating material.

In some embodiments, pharmaceutical agents or compositions of thepresent invention find particular use as vaccines. In the context of thepresent invention, the term “vaccine” (also referred to as an“immunogenic composition”) refers to an agent or composition that hasthe function to improve, enhance and/or induce anti-tumor immunity uponinoculation into animals.

Unless otherwise defined, the term “cancer” refers to cancers or tumorsthat overexpress the UBE2T gene, examples of which include, but are notlimited to, bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, esophageal cancer,gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor.

Unless otherwise defined, the terms “cytotoxic T lymphocyte”, “cytotoxicT cell” and “CTL” are used interchangeably herein and unless otherwisespecifically indicated, refer to a sub-group of T lymphocytes that arecapable of recognizing non-self cells (e.g., tumor/cancer cells,virus-infected cells) and inducing the death of such cells.

Unless otherwise defined, the term “HLA-A24”, as used herein,representatively refers to the subtypes, examples of which include, butare not limited to, HLA-A*2401, HLA-A*2402, HLA-A*2403, HLA-A*2404,HLA-A*2407, HLA-A*2408, HLA-A*2420, HLA-A*2425 and HLA-A*2488.

Unless otherwise defined, the term “HLA-A2”, as used herein,representatively refers to the subtypes , examples of which include, butare not limited to, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204,HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0213,HLA-A*0216, HLA-A*0218, HLA-A*0219, HLA-A*0228 and HLA-A*0250.

Unless otherwise defined, the term “kit” as used herein, is used inreference to a combination of reagents and other materials. It iscontemplated herein that the kit may include microarray, chip, marker,and so on. It is not intended that the term “kit” be limited to aparticular combination of reagents and/or materials.

As used herein, in the context of a subject or patient, the phrase“subject's (or patient's) HLA antigen is HLA-A24 or HLA-A2” refers tothat the subject or patient homozygously or heterozygously possessHLA-A24 or HLA-A2 antigen gene, and HLA-A24 or HLA-A2 antigen isexpressed in cells of the subject or patient as an HLA antigen.

To the extent that the methods and compositions of the present inventionfind utility in the context of the “treatment” of cancer, a treatment isdeemed “efficacious” if it leads to clinical benefit such as decrease insize, prevalence, or metastatic potential of cancer in a subject,prolongation of survival time, suppression of postoperative recurrenceand so on. When the treatment is applied prophylactically, “efficacious”means that it retards or prevents cancer from forming or prevents oralleviates a clinical symptom of cancer. Efficaciousness is determinedin association with any known method for diagnosing or treating theparticular tumor type.

To the extent that the methods and compositions of the present inventionfind utility in the context of the “prevention” and “prophylaxis” ofcancer, such terms are interchangeably used herein to refer to anyactivity that reduces the burden of mortality or morbidity from disease.Prevention and prophylaxis can occur “at primary, secondary and tertiaryprevention levels”. While primary prevention and prophylaxis avoid thedevelopment of a disease, secondary and tertiary levels of preventionand prophylaxis encompass activities aimed at the prevention andprophylaxis of the progression of a disease and the emergence ofsymptoms as well as reducing the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications. Alternatively, prevention and prophylaxis can include awide range of prophylactic therapies aimed at alleviating the severityof the particular disorder, e.g., reducing the proliferation andmetastasis of tumors.

In the context of the present invention, the treatment and/orprophylaxis of cancer and/or the prevention of postoperative recurrencethereof include any activity that leads to the following events, such asthe surgical removal of cancer cells, the inhibition of the growth ofcancerous cells, the involution or regression of a tumor, the inductionof remission and suppression of occurrence of cancer, the tumorregression, and the reduction or inhibition of metastasis, thesuppression of post operative recurrence of cancer, and prolongation ofsurvival time. Effective treatment and/or the prophylaxis of cancerdecreases mortality and improves the prognosis of individuals havingcancer, decreases the levels of tumor markers in the blood, andalleviates detectable symptoms accompanying cancer. For example,reduction or improvement of symptoms constitutes effectively treatingand/or the prophylaxis includes 10%, 20%, 30% or more reduction, orstable disease.

In the context of the present invention, the term “antibody” refers toimmunoglobulins and fragments thereof that are specifically reactive toa designated protein or peptide thereof. An antibody can include humanantibodies, primatized antibodies, chimeric antibodies, bispecificantibodies, humanized antibodies, antibodies fused to other proteins orradiolabels, and antibody fragments. Furthermore, an antibody herein isused in the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, andantibody fragments as long as they exhibit the desired biologicalactivity. An “antibody” indicates all classes (e.g., IgA, IgD, IgE, IgGand IgM).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

II. Peptides

Peptides of the present invention described in detail below may bereferred to as “UBE2T peptide(s) “or” UBE2T polypeptide(s)”.

To demonstrate that peptides derived from UBE2T function as an antigenrecognized by CTLs, peptides derived from UBE2T (SEQ ID NO: 65) wereanalyzed to determine whether they were antigen epitopes restricted byHLA-A24 or HLA-A2 which are commonly encountered HLA alleles (Date Y etal., Tissue Antigens 47: 93-101, 1996; Kondo A et al., J Immunol 155:4307-12, 1995; Kubo RT et al., J Immunol 152: 3913-24, 1994).

Candidates of HLA-A24 binding peptides derived from UBE2T wereidentified based on their binding affinities to HLA-A24. The followingcandidate peptides were identified: SEQ ID NOs: 1, 2 and 4 to 27.

Moreover, after in vitro stimulation of T-cells by dendritic cells (DCs)pulsed (loaded) with these peptides, CTLs were successfully establishedusing each of the following peptides:

-   UBE2T-A24-9-60 (SEQ ID NO: 1), UBE2T-A24-9-45 (SEQ ID NO: 2),-   UBE2T-A24-9-133 (SEQ ID NO: 4), UBE2T-A24-9-138 (SEQ ID NO: 6),-   UBE2T-A24-9-43 (SEQ ID NO: 11), UBE2T-A24-9-106 (SEQ ID NO: 12),-   UBE2T-A24-9-3 (SEQ ID NO: 13), UBE2T-A24-9-105 (SEQ ID NO: 15),-   UBE2T-A24-10-130 (SEQ ID NO: 17), UBE2T-A24-10-131 (SEQ ID NO: 19),-   UBE2T-A24-10-133 (SEQ ID NO: 20), UBE2T-A24-10-99 (SEQ ID NO: 21),-   UBE2T-A24-10-154 (SEQ ID NO: 22), UBE2T-A24-10-105 (SEQ ID NO: 23),-   UBE2T-A24-10-115 (SEQ ID NO: 24), UBE2T-A24-10-177 (SEQ ID NO: 25),    and-   UBE2T-A24-10-44 (SEQ ID NO: 27).

Candidates of HLA-A2 binding peptides derived from UBE2T were identifiedbased on their binding affinities to HLA-A2. The following peptides wereidentified: SEQ ID NOs: 29 to 63.

Moreover, after in vitro stimulation of T-cells by dendritic cells (DCs)pulsed (loaded) with these peptides, CTLs were successfully establishedusing each of the following peptides:

-   UBE2T-A02-9-107 (SEQ ID NO: 29), UBE2T-A02-9-30 (SEQ ID NO: 30),-   UBE2T-A02-9-106 (SEQ ID NO: 32), UBE2T-A02-9-49 (SEQ ID NO: 36),-   UBE2T-A02-9-13 (SEQ ID NO: 38), UBE2T-A02-9-132 (SEQ ID NO: 41),-   UBE2T-A02-10-70 (SEQ ID NO: 48), UBE2T-A02-10-6 (SEQ ID NO: 49),-   UBE2T-A02-10-106 (SEQ ID NO: 51), UBE2T-A02-10-102 (SEQ ID NO: 52),-   UBE2T-A02-10-30 (SEQ ID NO: 53), UBE2T-A02-10-101 (SEQ ID NO: 55),-   UBE2T-A02-10-29 (SEQ ID NO: 56) and UBE2T-A02-10-38 (SEQ ID NO: 58).

These established CTLs show potent specific CTL activity against targetcells pulsed with respective peptides. These results herein demonstratethat UBE2T is an antigen recognized by CTLs and that the above peptidesare epitope peptides of UBE2T restricted by HLA-A24 or HLA-A2.

Accordingly, in preferred embodiments, peptides having the amino acidsequence of SEQ ID NO: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22,23, 24, 25, or 27 may be used for the induction of CTLs in a subjectthat has been identified as having HLA-A24 prior to the induction.Likewise, peptides having the amino acid sequence of SEQ ID NO: 29, 30,32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58 may be used for theinduction of CTL in a subject that has been identified as having HLA-A2prior to the induction.

Since the UBE2T gene is over-expressed in cancer cells and tissues,including for example those of bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer, esophagealcancer, gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor, and not expressed in most normalorgans, it represents a good target for immunotherapy. Thus, the presentinvention provides nonapeptides (peptides composed of nine amino acidresidues) and decapeptides (peptides composed of ten amino acidresidues) corresponding to CTL-recognized epitopes from UBE2T.Alternatively, the present invention provides isolated peptides whichcan induce CTLs, wherein the peptide is composed of an immunologicallyactive fragment of UBE2T. In some embodiments, the present inventionprovides peptides including an amino acid sequence selected from amongSEQ TD NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25,27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58. Inpreferred embodiments, the peptides of the present invention arenonapeptides or decapeptides including an amino acid sequence selectedfrom among SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22,23, 24, 25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and58. The preferred examples of the peptides of the present inventioninclude peptides consisting of an amino acid sequence selected fromamong SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23,24, 25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58.

The peptides of the present invention, particularly the nonapeptides anddecapeptides of the present invention, may be flanked with additionalamino acid residues, as long as the resulting peptide retains its CTLinducibility. The particular additional amino acid residues may becomposed of any kind of amino acids, as long as they do not impair theCTL inducibility of the original peptide. Thus, the present inventionencompasses peptides having CTL inducibility, in particular peptidesderived from UBE2T (e.g., peptides including the amino acid sequence ofSEQ ID NO: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25,27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58). Suchpeptides are, for example, less than about 40 amino acids, often lessthan about 20 amino acids, and usually less than about 15 amino acids.More specifically, the size of such peptide is preferably in the rangeof 10 to 40 amino acids, such as in the range of 10 to 20 amino acids,for example in the range 10 to 15 amino acids.

Generally, it is known that the modification of one, two or severalamino acids in a peptide do not influence the function of the peptide,and in some cases even enhance the desired function of the originalpeptide. In fact, modified peptides (i.e., peptides composed of an aminoacid sequence in which 1, 2 or several amino acid residues have beenmodified (i.e., substituted, added, deleted and/or inserted) as comparedto an original reference sequence) have been known to retain thebiological activity of the original peptide (Mark et al., Proc Natl AcadSci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10:6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79:6409-13). Thus, in one embodiment, the peptides of the present inventionhave both CTL inducibility and an amino acid sequence selected fromamong SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23,24, 25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58,in which one, two or several amino acids are added, deleted, insertedand/or substituted. In other words, the peptides of the presentinvention have both CTL inducibility and an amino acid sequence in whichone, two or several amino acid(s) are substituted, deleted, insertedand/or added in the amino acid sequence selected from among SEQ ID NOs:1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30,32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58, provided the modifiedpeptides retain the CTL inducibility of the original peptide.

Those of skill in the art will recognize that individual modifications(i.e., deletions, insertions, additions and/or substitutions) to anamino acid sequence that alter a single amino acid or a small percentageof the overall amino acid sequence tend to result in the conservation ofthe properties of the original amino acid side-chain. As such, they areconventionally referred to as “conservative substitutions” or“conservative modifications”, wherein the alteration of a proteinresults in a protein with similar functions to the original protein.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Examples of amino acid side-chainscharacteristics that are desirable to conserve include, for example:hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic aminoacids (R, D, N, C, E, Q, G, H, K, S, T), and side-chains having thefollowing functional groups or characteristics in common: an aliphaticside-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain(S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acidand amide containing side-chain (D, N, E, Q); a base containingside-chain (R, K, H); and an aromatic containing side-chain (H, F, Y,W). In addition, the following eight groups each contain amino acidsthat are accepted in the art as conservative substitutions for oneanother:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins    1984).

Such conservatively modified peptides are also considered to be peptidesof the present invention. However, peptides of the present invention arenot restricted thereto and may include non-conservative modifications,as long as the resulting modified peptide retains the requisite CTLinducibility of the original unmodified peptide. Furthermore, themodified peptides should not exclude CTL inducible peptides derived frompolymorphic variants, interspecies homologues, and alleles of UBE2T.

Amino acid residues may be inserted, substituted and/or added to thepeptides of the present invention or, alternatively, amino acid residuesmay be deleted therefrom to achieve a higher binding affinity. To retainthe requisite CTL inducibility, one of skill in the art preferablymodifies (i.e., deletes, inserts, adds and/or substitutes) only a smallnumber (for example, 1, 2 or several) or a small percentage of aminoacids. Herein, the term “several” means 5 or fewer amino acids, forexample, 4 or 3 or fewer. The percentage of amino acids to be modifiedmay be, for example, 30% or less, preferably 20% or less, morepreferably 15% or less, and even more preferably 10% or less, forexample 1 to 5%.

When used in the context of cancer immunotherapy, the peptides of thepresent invention may be presented on the surface of a cell or exosomeas a complex with an HLA antigen. Therefore, it is preferable to selectpeptides that not only induce CTLs but also possess high bindingaffinity to the HLA antigen. To that end, the peptides can be modifiedby substitution, insertion, deletion and/or addition of the amino acidresidues to yield a modified peptide having improved binding affinity tothe HLA antigen. In addition to peptides that are naturally displayed,since the regularity of the sequences of peptides displayed by bindingto HLA antigens has already been known (Kubo R T et al., J Immunol 1994,152: 3913-24; Rammensee H G et al., Immunogenetics 1995, 41: 178-228;Kondo et al., J Immunol 1994, 155: 4307-12; Falk K, et al., Nature. 1991May 23;351(6324):290-6.), modifications based on such regularity may beintroduced into the immunogenic peptides of the present invention.

For example, peptides possessing high HLA-A24 binding affinity tend tohave the second amino acid from the N-terminus substituted withphenylalanine, tyrosine, methionine or tryptophan. Likewise, peptides inwhich the C-terminal amino acid is substituted with phenylalanine,leucine, isoleucine, tryptophan or methionine tend to have high HLA-A24binding affinity. Accordingly, it may be desirable to substitute thesecond amino acid from the N-terminus with phenylalanine, tyrosine,methionine or tryptophan, and/or the amino acid at the C-terminus withleucine, isoleucine, tryptophan or methionine in order to increase theHLA-A24 binding affinity. Thus, peptides having an amino acid sequenceselected from among SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20,21, 22, 23, 24, 25, and 27, in which the second amino acid from theN-terminus of the amino acid sequence of the SEQ ID NO is substitutedwith phenylalanine, tyrosine, methionine or tryptophan, and/or in whichthe C-terminus of the amino acid sequence of the SEQ ID NO issubstituted with leucine, isoleucine, tryptophan or methionine areencompassed by the present invention. Also, the present inventionencompasses the peptides including an amino acid sequence in which one,two or several amino acid are substituted, deleted, inserted and/oradded in the SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22,23, 24, 25, and 27, such peptides having one or both of the followingcharacteristic of (a) the second amino acid from the N-terminus isphenylalanine, tyrosine, methionine or tryptophan; and (b) theC-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophanor methionine. In preferred embodiments, the peptides of the presentinvention include an amino acid sequence in which the second amino acidfrom the N-terminus is substituted with phenylalanine, tyrosine,methionine or tryptophan, and/or the C-terminal amino acid issubstituted with phenylalanine, leucine, isoleucine, tryptophan ormethionine in the amino acid sequence of SEQ ID NOs: 1, 2, 4, 6, 11, 12,13, 15, 17, 19, 20, 21, 22, 23, 24, 25, and 27.

Likewise, peptides possessing high HLA-A2 binding affinity tend to havethe second amino acid from the N-terminus substituted with leucine ormethionine and/or the amino acid at the C-terminus substituted withvaline or leucine. Accordingly, it may be desirable to substitute thesecond amino acid from the N-terminus with leucine or methionine, and/orthe amino acid at the C-terminus with valine or leucine in order toincrease the HLA-A2 binding affinity. Thus, peptides having an aminoacid sequence selected from among SEQ ID NOs: 29, 30, 32, 36, 38, 41,48, 49, 51, 52, 53, 55, 56 and 58, in which the second amino acid fromthe N-terminus of the amino acid sequence of the SEQ ID NO issubstituted with leucine or methionine, and/or wherein the C-terminus ofthe amino acid sequence of the SEQ ID NO is substituted with valine orleucine are encompassed by the present invention. Also, the presentinvention encompasses the peptides including an amino acid sequence inwhich one, two or several amino acid are substituted, deleted, insertedand/or added in the SEQ ID NOs: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52,53, 55, 56 and 58, such peptides having one or both of the followingcharacteristic of (a) the second amino acid from the N-terminus isleucine or methionine; and (b) the C-terminal amino acid is valine orleucine. In preferred embodiments, the peptides of the present inventioninclude an amino acid sequence in which the second amino acid from theN-terminus is substituted with leucine or methionine, and/or theC-terminal amino acid is substituted with valine or leucine in the aminoacid sequence of SEQ ID NOs: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53,55, 56 and 58.

Substitutions can be introduced not only at the terminal amino acids butalso at the positions of potential T cell receptor (TCR) recognitionsites of peptides. Several studies have demonstrated that a peptide withamino acid substitutions may have equal to or better function than thatof the original, for example, CAP1, p53₍₂₆₄₋₂₇₂₎, Her-2/neu₍₃₆₉₋₃₇₇₎ orgp 100₍₂₀₉₋₂₁₇₎ (Zaremba et al. Cancer Res. 57, 4570-4577, 1997,Hoffmann T K et al. J Immunol. (2002);168(3):1338-47., Dionne S O et al.Cancer Immunol immunother. (2003) 52: 199-206 and Dionne S O et al.Cancer Immunology, Immunotherapy (2004) 53, 307-314).

The present invention also contemplates the addition of 1, 2 or severalamino acids can also be added to the N and/or C-terminus of the peptidesof the present invention. Such modified peptides having CTL inducibilityare also included in the present invention.

For example, the present invention provides an isolated peptide of lessthan 15, 14, 13, 12, 11, or 10 amino acids in length, which has CTLinducibility and comprises the amino acid sequence selected from thegroup consisting of:

(i) an amino acid sequence in which 1, 2 or several amino acid(s) aremodified in the amino acid sequence selected from the group consistingof SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, and 15,

(ii) the amino acid sequence of (i), wherein the amino acid sequence hasone or both of the following characteristics:

(a) the second amino acid from the N-terminus of said SEQ ID NOs is oris modified to be an amino acid selected from the group consisting ofphenylalanine, tyrosine, methionine and tryptophan, and

(b) the C-terminal amino acid of said SEQ ID NOs is or is modified to bean amino acid selected from the group consisting of phenylalanine,leucine, isoleucine, tryptophan and methionine, and

(iii) the amino acid sequence in which 1, 2 or several amino acid(s) aremodified in the amino acid sequence selected from the group consistingof SEQ ID NOs: ,29, 30, 32, 36, 38, and 41

(iv) the amino acid sequence of (iii), wherein the amino acid sequencehas one or both of the following characteristics:

(a) the second amino acid from the N-terminus of said SEQ ID NO is or ismodified to be an amino acid selected from the group consisting ofleucine and methionine; and

(b) the C-terminal amino acid of said SEQ ID NO is or is modified to bean amino acid selected from the group consisting of valine and leucine.

Moreover, the present invention also provides an isolated peptide ofless than 15, 14, 13, 12, or 11 amino acids in length, which has CTLinducibility and comprises the amino acid sequence selected from thegroup consisting of:

(i′) an amino acid sequence in which 1, 2 or several amino acid(s) aremodified in the amino acid sequence selected from the group consistingof SEQ ID NOs: 17, 19, 20, 21, 22, 23, 24, 25, and 27,

(ii′) the amino acid sequence of (i′), wherein the amino acid sequencehas one or both of the following characteristics:

(a) the second amino acid from the N-terminus of said SEQ ID NOs is oris modified to be an amino acid selected from the group consisting ofphenylalanine, tyrosine, methionine and tryptophan, and

(b) the C-terminal amino acid of said SEQ ID NOs is or is modified to bean amino acid selected from the group consisting of phenylalanine,leucine, isoleucine, tryptophan, and methionine,

(iii′) an amino acid sequence in which 1, 2 or several amino acid(s) aremodified in the amino acid sequence selected from the group consistingof SEQ ID NOs: 48, 49, 51, 52, 53, 55, 56 and 58,

(iv′) the amino acid sequence of (iii′), wherein the amino acid sequencehas one or both of the following characteristics:

(a) the second amino acid from the N-terminus of said SEQ ID NOs is oris modified to be an amino acid selected from the group consisting ofleucine and methionine; and

(b) the C-terminal amino acid of said SEQ ID NOs is or is modified to bean amino acid selected from the group consisting of valine and leucine.

These peptides are processed in an APC to present a peptide selectedfrom the group consisting of (i) to (iv) and (i′) to (iv′) thereon, whenthese peptides are contacted with, or introduced in APC.

However, when the peptide sequence is identical to a portion of theamino acid sequence of an endogenous or exogenous protein having adifferent function, side effects such as autoimmune disorders and/orallergic symptoms against specific substances may be induced. Therefore,it is preferable to first perform homology searches using availabledatabases to avoid situations in which the sequence of the peptidematches the amino acid sequence of another protein. When it becomesclear from the homology searches that there exists not even a peptidewith 1 or 2 amino acid differences as compared to the objective peptide,the objective peptide can be modified in order to increase its bindingaffinity with HLA antigens, and/or increase its CTL inducibility withoutany danger of such side effects.

Although peptides having high binding affinity to the HLA antigens areexpected to be effective, the candidate peptides, which are selectedaccording to the presence of high binding affinity as an indicator, arefurther examined for the presence of CTL inducibility. Herein, thephrase “CTL inducibility” indicates the ability of a peptide to induce acytotoxic T lymphocyte (CTL) when presented on an antigen-presentingcell (APC). Further, “CTL inducibility” includes the ability of apeptide to induce CTL activation, CTL proliferation, promote lysis oftarget cells by a CTL, and to increase IFN-gamma production by a CTL.

Confirmation of CTL inducibility is accomplished by inducing APCscarrying human MHC antigens (for example, B-lymphocytes, macrophages,and dendritic cells (DCs)), or more specifically DCs derived from humanperipheral blood mononuclear leukocytes, and after stimulation of APCswith a test peptides, mixing the APCs with CD8 positive T cells toinduce CTLs, and then measuring the IFN-gamma against the target cellsproduced and released by CTLs. As the reaction system, transgenicanimals that have been produced to express a human HLA antigen (forexample, those described in BenMohamed L et al., Hum Immunol 2000,61(8): 764-79, Related Articles, Books, Linkout Induction of CTLresponse by a minimal epitope vaccine in HLA-A*0201/DR1 transgenic mice:dependence on HLA class II restricted T(H) response) can be used.Alternatively, the target cells may be radiolabeled with ⁵¹Cr and such,and cytotoxic activity of CTLs may be calculated from radioactivityreleased from the target cells. Alternatively, CTL inducibility can beassessed by measuring IFN-gamma produced and released by CTLs in thepresence of cells that carry immobilized peptides, and visualizing theinhibition zone on the media using anti-IFN-gamma monoclonal antibodies.

In addition to the above-described modifications, the peptides of thepresent invention can also be linked to other peptides, as long as theresulting linked peptide retains the requisite CTL inducibility of theoriginal peptide, and more preferably also retains the requisite HLAbinding ability thereof. Examples of suitable “other” peptides include:the peptides of the present invention or the CTL-inducible peptidesderived from other TAAs. The peptide of the present invention can belinked to “another” peptide directly or indirectly via a linker. Thelinkers between the peptides are well known in the art and include, forexample AAY (Daftarian P M, et al., J Trans Med 2007, 5:26), AAA, NKRK(Sutmuller R P, et al., J Immunol. 2000, 165: 7308-7315) or K (Ota S, etal., Can Res. 62, 1471-1476, Kawamura K S, et al., J Immunol. 2002, 168:5709-5715).

The above described linked peptides are referred to herein as“polytopes”, i.e., groups of two or more potentially immunogenic orimmune response stimulating peptides which can be joined together invarious arrangements (e.g., concatenated, overlapping). The polytope (ornucleic acid encoding the polytope) can be administered in accordancewith a standard immunization protocol, e.g., to animals, to test theeffectiveness of the polytope in stimulating, enhancing and/or provokingan immune response.

The peptides can be joined together directly or via the use of flankingsequences to form polytopes, and the use of polytopes as vaccines iswell known in the art (see, e.g., Thomson et al., Proc. Natl. Acad. SciUSA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol.15(12):1280-1284, 1997; Thomson et al., J Immunol. 157(2):822-826, 1996;Tarn et al., J Exp. Med. 171(1):299-306, 1990). Polytopes containingvarious numbers and combinations of epitopes can be prepared and testedfor recognition by CTLs and for efficacy in increasing an immuneresponse.

The peptides of the present invention may also be linked to othersubstances, as long as the resulting linked peptide retains therequisite CTL inducibility of the original peptide. Examples of suitablesubstances include, for example: peptides, lipids, sugar and sugarchains, acetyl groups, natural and synthetic polymers, etc. The peptidesmay contain modifications such as glycosylation, side chain oxidation,or phosphorylation, etc., provided the modifications do not destroy thebiological activity of the original peptide. These kinds ofmoditications may be performed to confer additional functions (e.g.,targeting function, and delivery function) or to stabilize the peptide.

For example, to increase the in vivo stability of a peptide, it is knownin the art to introduce D-amino acids, amino acid mimetics or unnaturalamino acids; this concept can also be adapted to the peptides of thepresent invention. The stability of a peptide can be assayed in a numberof ways. For instance, peptidases and various biological media, such ashuman plasma and serum, can be used to test stability (see, e.g.,Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

Moreover, as noted above, among the modified peptides in which aresubstituted, deleted inserted and/or added by 1, 2 or several amino acidresidues, those having same or higher activity as compared to originalpeptides can be screened for or selected. The present invention,therefore, also provides the method of screening for or selectingmodified peptides having same or higher activity as compared tooriginals. An illustrative method includes the steps of:

a: substituting, deleting, inserting and/or adding at least one aminoacid residue of a peptide of the present invention,

b: determining the activity of the peptide modified in step a, and

c: selecting the peptide having same or higher activity as compared tothe original peptide.

Preferably, the activity of the peptide to be assayed is CTLinducibility.

In preferred embodiments, the present invention provides a method ofscreening for a peptide having an ability to induce a CTL that hasspecific cytotoxic activity against a cell that presents a fragmentderived from UBE2T, wherein the method comprises the steps of:

(i) providing a candidate sequence consisting of an amino acid sequencemodified by substituting, deleting, inserting and/or adding one, two orseveral amino acid residues to an original amino acid sequence, whereinthe original amino acid sequence is selected from the group consistingof SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24,25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58;

(ii) selecting a candidate sequence that does not have substantialsignificant homology (or sequence identity) with the peptides derivedfrom any known human gene products other than UBE2T;

(iii) contacting a peptide consisting of the candidate sequence selectedin step (ii) with an antigen presenting cell;

(iv) contacting the antigen presenting cell of step (iii) with a CD8positive T cell; and

(v) identifying the peptide of which CTL inducibility is same to orhigher than a peptide consisting of the original amino acid sequence.

III. Preparation of UBE2T Peptides

The peptides of the present invention can be prepared using well knowntechniques. For example, the peptides can be prepared synthetically,using recombinant DNA technology or chemical synthesis. The peptides ofthe present invention can be synthesized individually or as longerpolypeptides including two or more peptides. The peptides can then beisolated i.e., purified or isolated so as to be substantially free ofother naturally occurring host cell proteins and fragments thereof, orany other chemical substances.

The peptides of the present invention may contain modifications, such asglycosylation, side chain oxidation, or phosphorylation, provided themodifications do not destroy the biological activity of the originalpeptide. Other illustrative modifications include incorporation of oneor more D-amino acids or other amino acid mimetics that can be used, forexample, to increase the serum half life of the peptides.

Peptides of the present invention can be obtained through chemicalsynthesis based on the selected amino acid sequence. For example,conventional peptide synthesis methods that can be adopted for thesynthesis include:

(i) Peptide Synthesis, Interscience, New York, 1966;

(ii) The Proteins, Vol. 2, Academic Press, New York, 1976;

(iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975;

(iv) Basics and Experiment of Peptide Synthesis (in Japanese), MaruzenCo., 1985;

(v) Development of Pharmaceuticals (second volume) (in Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991;

(vi) WO99/67288; and

(vii) Barany G. & Merrifield R. B., Peptides Vol. 2, “Solid PhasePeptide Synthesis”, Academic Press, New York, 1980, 100-118.

Alternatively, the peptides of the present invention can be obtainedadopting any known genetic engineering method for producing peptides(e.g. Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss &Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). Forexample, first, a suitable vector harboring a polynucleotide encodingthe objective peptide in an expressible form (e.g. downstream of aregulatory sequence corresponding to a promoter sequence) is preparedand transformed into a suitable host cell. Such vectors and host cellsare also provided by the present invention. The host cell is thencultured to produce the peptide of interest. The peptide can also beproduced in vitro adopting an in vitro translation system.

IV. Polynucleotides

The present invention also provides polynucleotides that encode any ofthe aforementioned peptides of the present invention. These includepolynucleotides derived from the natural occurring UBE2T gene (e.g.,GenBank Accession No. NM_014176 (SEQ ID NO: 64)) as well as those havinga conservatively modified nucleotide sequence thereof. Herein, thephrase “conservatively modified nucleotide sequence” refers to sequenceswhich encode identical or essentially identical amino acid sequences.Due to the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG, and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a peptide also describes every possible silent variationof the nucleic acid. One of ordinary skill in the art will recognizethat each codon in a nucleic acid (except AUG, which is ordinarily theonly codon for methionine, and TGG, which is ordinarily the only codonfor tryptophan) can be modified to yield a functionally identicalmolecule. Accordingly, each silent variation of a nucleic acid thatencodes a peptide is implicitly described in each disclosed sequence.

The polynucleotide of the present invention can be composed of DNA, RNA,and derivatives thereof. As is well known in the art, a DNA is suitablycomposed of bases such as A, T, C, and G, and T is replaced by U in anRNA. One of skill in the art will recognize that non-naturally occurringbases may be included in polynucleotides, as well.

The polynucleotide of the present invention can encode multiple peptidesof the present invention with or without intervening amino acidsequences. For example, the intervening amino acid sequence can providea cleavage site (e.g., enzyme recognition sequence) of thepolynucleotide or the translated peptides. Furthermore, thepolynucleotide of the present invention can include any additionalsequences to the coding sequence encoding the peptide of the presentinvention. For example, the polynucleotide of the present invention canbe a recombinant polynucleotide that includes regulatory sequencesrequired for the expression of the peptide or can be an expressionvector (plasmid) with marker genes and such. In general, suchrecombinant polynucleotides can be prepared by the manipulation ofpolynucleotides through conventional recombinant techniques using, forexample, polymerases and endonucleases.

Both recombinant and chemical synthesis techniques can be used toproduce the polynucleotides of the present invention. For example, thepolynucleotide of the present invention can be produced by insertioninto an appropriate vector, which can be expressed when transfected intoa competent cell. Alternatively, the polynucleotide of the presentinvention can be amplified using PCR techniques or expression insuitable hosts (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1989).Alternatively, the polynucleotide of the present invention can besynthesized using the solid phase techniques, as described in Beaucage SL & Iyer R P, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J1984, 3: 801-5.

V. Exosomes

The present invention further provides intracellular vesicles calledexosomes, which present complexes formed between the peptides of thepresent invention and HLA antigens on their surface. Exosomes can beprepared, for example, using the methods detailed in Japanese PatentPublication No. H11-510507 and WO99/03499, and can be prepared usingAPCs obtained from patients who are subject to treatment and/orprevention. The exosomes of the present invention can be inoculated asvaccines, in a fashion similar to the peptides of the present invention.

The type of HLA antigens included in the complexes must match that ofthe subject requiring treatment and/or prevention. For example, in theJapanese population, HLA-A24 and HLA-A2, particularly HLA-A*2402 andHLA-A*0201 and HLA-A*0206, are prevalent and therefore would beappropriate for treatment of Japanese patients. The use of the HLA-A24or HLA-A2 type that are highly expressed among the Japanese andCaucasian is favorable for obtaining effective results, and subtypessuch as HLA-A*2402, HLA-A*0201 and HLA-A*0206 also find use. Typically,in the clinic, the type of HLA antigen of the patient requiringtreatment is investigated in advance, which enables the appropriateselection of peptides having high levels of binding affinity to theparticular antigen, or having CTL inducibility by antigen presentation.Furthermore, in order to obtain peptides having both high bindingaffinity and CTL inducibility, substitution, insertion, deletion and/oraddition of 1, 2, or several amino acids can be performed based on theamino acid sequence of the naturally occurring UBE2T partial peptide.

When using the HLA-A24 type of HLA antigen for the exosome of thepresent invention, peptides having an amino acid sequence selected fromamong SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23,24, 25 and 27 have particular utility.

Alternatively, when using the HLA-A2 type of HLA antigen for the exosomeof the present invention, peptides having an amino acid sequenceselected from among SEQ ID NOs: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52,53, 55, 56 and 58 have particular utility.

In some embodiments, the exosomes of the present invention present acomplex of the peptide of the present invention and HLA-A24 or HLA-A2antigen on their surface. In typical embodiments, the exosome of thepresent invention presents a complex of a peptide having an amino acidsequence of SEQ ID NO: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22,23, 24, 25 or 27 (or modified peptide thereof) and HLA-A24 on itssurface. In other embodiments, the exosome of the present inventionpresents a complex of a peptide having an amino acid sequence of SEQ IDNO: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58 (ormodified peptide thereof) and HLA-A2 on its surface.

VI. Antigen-Presenting Cells (APCs)

The present invention also provides isolated antigen-presenting cells(APCs) that present complexes formed between HLA antigens and thepeptides of the present invention on their surface. The APCs can bederived from patients who are subject to treatment and/or prevention,and can be administered as vaccines by themselves or in combination withother drugs including the peptides, exosomes, or CTLs of the presentinvention.

The APCs are not limited to a particular kind of cells and includedendritic cells (DCs), Langerhans cells, macrophages, B cells, andactivated T cells, which are known to present proteinaceous antigens ontheir cell surface so as to be recognized by lymphocytes. Since DCs arerepresentative APCs having the strongest CTL inducing activity amongAPCs, DCs are suitable for the APCs of the present invention.

For example, the APCs of the present invention can be obtained byinducing DCs from peripheral blood monocytes and then contacting(stimulating) them with the peptides of the present invention in vitro,ex vivo or in vivo. When the peptides of the present invention areadministered to a subject, APCs that present the peptides of the presentinvention are induced in the body of the subject. Therefore, the APCs ofthe present invention can be obtained by collecting the APCs from asubject after administering the peptides of the present invention to thesubject. Alternatively, the APCs of the present invention can beobtained by contacting APCs, which have been collected from a subject,with the peptide of the present invention.

The APCs of the present invention can be administered to a subject forinducing immune response against cancer in the subject by themselves orin combination with other drugs including the peptides, exosomes or CTLsof the present invention. For example, the ex vivo administration caninclude steps of:

a: collecting APCs from a first subject,

b: contacting the APCs of step a, with the peptide of the presentinvention, and

c: administering the APCs of step b to a second subject.

The first subject and the second subject can be the same individual, ormay be different individuals. The APCs obtained by step b can beformulated and administered a vaccine for treating and/or preventingcancer, such as bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, esophageal cancer,gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor, but not limited thereto.

In the context of the present invention, one may utilize the peptides ofthe present invention for manufacturing a pharmaceutical compositioncapable of inducing an antigen-presenting cell. The present inventionalso provides a method or process for manufacturing a pharmaceuticalcomposition for inducing an antigen-presenting cell wherein the methodincludes the step of admixing or formulating the peptide of theinvention with a pharmaceutically acceptable carrier.

The present invention also provides for the use of the peptides of thepresent invention for inducing antigen-presenting cells.

According to an aspect of the present invention, the APCs of the presentinvention have CTL inducibility. In the context of the APCs, the phrase“CTL inducibility” refers to the ability of an APC to induce a CTL whencontacted with a CD8 positive T cell. Further, “CTL inducibility”includes the ability of an APC to induce CTL activation, CTLproliferation, promote lysis of a target cell by a CTL, and to increaseIFN-gamma production by a CTL. In particular, the APCs of the presentinvention have an ability to induce CTLs specific to UBE2T. Such APCshaving CTL inducibility can be prepared by a method that includes thestep of transferring a polynucleotide encoding the peptide of thepresent invention to APCs in vitro as well as the method mentionedabove. The introduced gene can be in the form of DNA or RNA. Examples ofmethods for introduction include, without particular limitations,various methods conventionally performed in this field, such aslipofection, electroporation, and calcium phosphate method can be used.More specifically, it can be performed as described in Reeves M E etal., Cancer Res 1996, 56: 5672-7; Butterfield L H et al., J Immunol1998, 161: 5607-13; Boczkowski D et al., J Exp Med 1996, 184: 465-72;Japanese Patent Publication No.JP2000-509281. By transferring the geneinto APCs, the gene undergoes transcription, translation, and such inthe cell, and then the obtained protein is processed by MHC Class I orClass II, and proceeds through a presentation pathway to present partialpeptides.

In some embodiments, the APCs of the present invention present complexesof HLA-A24 or HLA-A2 antigen and the peptide of the present invention ontheir surface. In typical embodiments, the APC of the present inventionpresents a complex of a peptide having an amino acid sequence of SEQ IDNO: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, or 27(or modified peptide thereof) and HLA-A24 on its surface. In otherembodiments, the APC of the present invention presents a complex of apeptide having an amino acid sequence of SEQ ID NO: 29, 30, 32, 36, 38,41, 48, 49, 51, 52, 53, 55, 56 or 58 (or modified peptide thereof) andHLA-A2 on its surface.

VII. Cytotoxic T Lymphocytes (CTLs)

A CTL induced against any one of the peptides of the present inventionstrengthens the immune response targeting cancer cells in vivo and thuscan be used as vaccines, in a fashion similar to the peptides per se.Thus, the present invention provides isolated CTLs that are specificallyinduced or activated by any one of the peptides of the presentinvention.

Such CTLs can be obtained by (1) administering the peptide(s) of thepresent invention to a subject, (2) contacting (stimulating)subject-derived APCs, and CD8 positive T cells, or peripheral bloodmononuclear leukocytes in vitro with the peptide(s) of the presentinvention, (3) contacting CD8 positive T cells or peripheral bloodmononuclear leukocytes in vitro with the APCs or exosomes presenting acomplex of an HLA antigen and the peptide on its surface or (4)introducing into a CD8 positive T cell a polynucleotide encoding both ofT cell receptor (TCR) subunits or polynucleotides encoding each of TCRsubunits, wherein the TCR formed by such subunits can bind a complex ofthe peptide of the present invention and HLA antigen on a cell surface.Such APCs or exosomes can be prepared by the methods described above.Details of the method of (4) are described bellow in section “VIII. TCell Receptor (TCR)”.

The CTLs of the present invention can be derived from patients who aresubject to treatment and/or prevention, and can be administered bythemselves or in combination with other drugs including the peptides,APCs or exosomes of the present invention for the purpose of regulatingeffects. The obtained CTLs act specifically against target cellspresenting the peptides of the present invention, for example, the samepeptides used for induction. The target cells can be cells thatendogenously express UBE2T, such as cancer cells, or cells that aretransfected with the UBE2T gene; and cells that present a peptide of thepresent invention on the cell surface due to stimulation by the peptidecan also serve as targets of activated CTL attack.

In some embodiments, the CTLs of the present invention are CTLs thatrecognize cells presenting complexes of an HLA-A24 or HLA-A2 antigen andthe peptide of the present invention. In the context of CTLs, the phrase“recognize a cell” refers to binding a complex of an HLA-A24 or HLA-A2antigen and the peptide of the present invention on the cell surface viaits TCR and showing specific cytotoxic activity against the cell.Herein, “specific cytotoxic activity” refers to showing cytotoxicactivity against a cell presenting a complex of an HLA-A24 or HLA-A2antigen and the peptide of the present invention but not other cells.Accordingly, the CTLs that show specific cytotoxic activity against acell presenting the peptide of the present invention are included in thepresent invention.

In typical embodiments, the CTL of the present invention can recognize acell presenting a peptide having an amino acid sequence of SEQ ID NO: 1,2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25 or 27 (ormodified peptide thereof) via an HLA-A24. In preferred embodiments, suchCTL of the present invention can recognize a cell expressing UBE2T andan HLA-A24 (e.g., HLA-A24 positive cancer cell) and show cytotoxicactivity against such cell.

In other embodiments, the CTL of the present invention can recognize acell presenting a peptide having an amino acid sequence of SEQ ID NO:29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58 (or modifiedpeptide thereof) via an HLA-A2. In preferred embodiments, such CTL ofthe present invention can recognize a cell expressing UBE2T and anHLA-A2 (e.g., HLA-A2 positive cancer cell) and show cytotoxic activityagainst such cell.

VIII. T Cell Receptor (TCR)

The present invention also provides a composition including apolynucleotide encoding both of TCR subunits or polynucleotides encodingeach of TCR subunits, wherein the TCR formed by such subunits can bindto a complex of an HLA antigen and the peptide of the present inventionon a cell surface, and methods of using the same. The TCR subunits havethe ability to form TCRs that confer specificity to T cells againsttumor cells expressing UBE2T. By using known methods in the art, thepolynucleotides encoding each of alpha- and beta- chains of the TCRsubunits of the CTL induced with one or more peptides of the presentinvention can be identified (WO2007/032255 and Morgan et al., J Immunol,171, 3288 (2003)). For example, the PCR method is preferred to analyzethe TCR. The PCR primers for the analysis can be, for example, 5′-Rprimers (5′-gtctaccaggcattcgcttcat-3′) as 5′ side primers (SEQ ID NO:66) and 3-TRa-C primers (5′-tcagctggaccacagccgcagcgt-3′) specific to TCRalpha chain C region (SEQ ID NO: 67), 3-TRb-C1 primers(5′-tcagaaatcctttctcttgac-3′) specific to TCR beta chain C1 region (SEQID NO: 68) or 3-TRbeta-C2 primers (5′ctagcctctggaatcctttctctt-3′)specific to TCR beta chain C2 region (SEQ ID NO: 69) as 3′ side primers,but not limited thereto. The derivative TCRs can bind target cellspresenting the peptide of the present invention with high avidity, andoptionally mediate efficient killing of target cells presenting thepeptide of the present invention in vivo and in vitro.

The polynucleotide encoding both of the TCR subunits or polynucleotidesencoding each of the TCR subunits can be incorporated into suitablevectors, e.g., retroviral vectors. These vectors are well known in theart. The polynucleotides or the vectors including them usefully can betransferred into a T cell (e.g., CD8 positive T cell), for example, a Tcell from a patient. Advantageously, the present invention provides anoff-the-shelf composition allowing rapid modification of a patient's ownT cells (or those of another mammal) to rapidly and easily producemodified T cells having excellent cancer cell killing properties.

The specific TCR against the peptide of the present invention is areceptor capable of specifically recognizing a complex of a peptide ofthe present invention and an HLA molecule, giving a T cell specificactivity against a target cell presenting a complex of the peptide ofthe present invention and an HLA antigen when the TCR is presented onthe surface of the T cell. A specific recognition of the above complexmay be confirmed by any known methods, preferred examples of whichinclude HLA multimer staining analysis using HLA molecules and peptidesof the present invention, and ELISPOT assay. By performing the ELISPOTassay, it can be confirmed that a T cell expressing the TCR on the cellsurface recognizes a cell by the TCR, and that signals are transmittedintracellularly. The confirmation that the above-mentioned TCR can givea T cell cytotoxic activity when the TCR exists on the T cell surfacemay also be carried out by a known method. A preferred method includes,for example, the determination of cytotoxic activity against a targetcell, such as chromium release assay.

Also, the present invention provides CTLs which are prepared bytransduction with the polynucleotides encoding both of the TCR subunitsor polynucleotides encoding each of the TCR subunits wherein the TCRformed by such TCR subunits can bind to the UBE2T peptide, e.g., apeptide having the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 11, 12,13, 15, 17, 19, 20, 21, 22, 23, 24, 25 or 27, in the context of HLA-A24,and also a peptide having the amino acid sequence of SEQ ID NO: 29, 30,32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58, in the context ofHLA-A2.

The transduced CTLs are capable of homing to cancer cells in vivo, andcan be expanded by well known culturing methods in vitro (e.g., Kawakamiet al., J Immunol., 142, 3452-3461 (1989)). The CTLs of the presentinvention can be used to form an immunogenic composition useful ineither or both of treatment and the prevention of cancer in a patient inneed of therapy or protection (See, WO2006/031221, the contents of whichare incorporated by reference herein).

IX. Pharmaceutical Agents or Compositions

The present invention also provides pharmaceutical agents orcompositions including at least one active ingredient selected fromamong:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC of the present invention;

(d) an exosome of the present invention; and

(e) a CTL of the present invention.

Since UBE2T expression is specifically elevated in cancers, examples ofwhich include, but are not limited to, bladder cancer, breast cancer,cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer,esophageal cancer, gastric cancer, diffuse-type gastric cancer, NSCLC,lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, SCLC, soft tissue tumor and testicular tumor, the peptides orpolynucleotides of the present invention may be used for the treatmentand/or prophylaxis of cancer, and/or for the prevention of apostoperative recurrence thereof. Thus, the present invention provides apharmaceutical composition or agent formulated for the treatment and/orprophylaxis of cancer, and/or for the prevention of a postoperativerecurrence thereof, such composition or agent including at least one ofthe peptides or polynucleotides of the present invention as an activeingredient. Alternatively, the peptides of the present invention can beexpressed on the surface of any of the foregoing exosomes or cells, suchas APCs for the use as pharmaceutical compositions or agents. Inaddition, the aforementioned CTLs which target any one of the peptidesof the present invention can also be used as the active ingredient ofthe pharmaceutical compositions or agents of the present invention.

Accordingly, the present invention provides agents or compositionsincluding at least one active ingredient selected from among:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC of the present invention;

(d) an exosome of the present invention; and

(e) a CTL of the present invention.

In the pharmaceutical agent or composition, such active ingredient ispresent in a therapeutically or pharmaceutically effective amount.

The pharmaceutical composition or agent of the present invention alsofind use as a vaccine. In the context of the present invention, thephrase “vaccine” (also referred to as an “immunogenic composition”)refers to an agent or composition that has the function to improve,enhance and/or induce anti-tumor immunity upon inoculation into ananimal. In other words, the present invention provides thepharmaceutical agents or compositions for inducing an immune responseagainst cancer in a subject. The amount of the peptide in such agent orcomposition may be an amount that is effective in significantlyenhancing or stimulating immunological response in a subject carrying acancer expressing UBE2T.

The pharmaceutical compositions or agents of the present invention canbe used to treat and/or prevent cancers, and/or prevent a postoperativerecurrence thereof in subjects or patients including human and any othermammals including, but not limited to, mice, rats, guinea-pigs, rabbits,cats, dogs, sheep, goats, pigs, cattle, horses, monkeys, baboons, andchimpanzees, particularly commercially important animals or domesticatedanimals. In some embodiments, the pharmaceutical agents or compositionsof the present invention can be formulated for the administration to asubject whose HLA antigen is HLA-A24 or HLA-A2.

In another embodiment, the present invention also provides the use of anactive ingredient in manufacturing a pharmaceutical composition or agentfor treating and/or preventing cancer or tumor, and/or preventing apost-operative recurrence thereof, said active ingredient selected fromamong:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC presenting a peptide of the present invention on its surface;

(d) an exosome presenting a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

Alternatively, the present invention further provides an activeingredient for use in the treatment and/or prevention of cancers ortumors, and/or prevention of a postoperative recurrence thereof, saidactive ingredient selected from among:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC presenting a peptide of the present invention on its surface;

(d) an exosome presenting a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

Alternatively, the present invention further provides a method orprocess for manufacturing a pharmaceutical composition or agent fortreating and/or preventing cancer or tumor, and/or preventing apost-operative recurrence thereof, wherein the method or processincludes the step of formulating a pharmaceutically or physiologicallyacceptable carrier with an active ingredient selected from among:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC presenting a peptide of the present invention on its surface;

(d) an exosome presenting a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

In another embodiment, the present invention also provides a method orprocess for manufacturing a pharmaceutical composition or agent fortreating and/or preventing cancer or tumor, and/or preventing apost-operative recurrence thereof, wherein the method or processincludes the steps of admixing an active ingredient with apharmaceutically or physiologically acceptable carrier, wherein theactive ingredient is selected from among:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC presenting a peptide of the present invention on its surface;

(d) an exosome presenting a peptide of the present invention on itssurface; and

(e) a CTL T cell of the present invention.

In another embodiment, the present invention also provides a method fortreating and/or preventing cancer or tumor, and/or preventing apost-operative recurrence thereof, wherein the method comprises the stepof administering to a subject at least one active ingredient selectedfrom among:

(a) a peptide of the present invention;

(b) a polynucleotide encoding such a peptide of the present invention inan expressible form;

(c) an APC presenting a peptide of the present invention on its surface;

(d) an exosome presenting a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

According to the present invention, peptides having an amino acidsequence selected from among SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17,19, 20, 21, 22, 23, 24, 25 and 27 have been found to be HLA-A24restricted epitope peptides that can induce potent and specific immuneresponse against cancer expressing HLA-A24 and UBE2T in a subject. Also,peptides having an amino acid sequence selected from among SEQ ID NOs:29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58 have beenfound to be HLA-A2 restricted epitope peptides that can induce potentand specific immune response against cancer expressing HLA-A2 and UBE2Tin a subject. Therefore, the pharmaceutical compositions or agents whichinclude any of these peptides with the amino acid sequence selected fromamong SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23,24, 25 and 27 are particularly suited for the administration to subjectswhose HLA antigen is HLA-A24. On the other hand, the pharmaceuticalcompositions or agents which include any of these peptides with theamino acid sequence selected from among SEQ ID NOs: 29, 30, 32, 36, 38,41, 48, 49, 51, 52, 53, 55, 56 and 58 are particularly suited for theadministration to subjects whose HLA antigen is HLA-A2. The same appliesto pharmaceutical compositions or agents that contain polynucleotidesencoding any of these peptides (i.e., the polynucleotides of the presentinvention).

Cancers to be treated and/or prevented by the pharmaceuticalcompositions or agents of the present invention are not limited andinclude all kinds of cancers in which UBE2T is involved, examples ofwhich include, but not limited to, bladder cancer, breast cancer,cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer,esophageal cancer, gastric cancer, diffuse-type gastric cancer, NSCLC,lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, SCLC, soft tissue tumor and testicular tumor.

The pharmaceutical compositions or agents of the present invention cancontain in addition to the aforementioned active ingredients, otherpeptides which have the ability to induce CTLs against cancerous cells,other polynucleotides encoding the other peptides, other cells thatpresent the other peptides, or such. Examples of such “other” peptidesthat have the ability to induce CTLs against cancerous cells include,but are not limited to peptides derived from cancer specific antigens(e.g., identified TAAs).

If needed, the pharmaceutical compositions or agents of the presentinvention can optionally include other therapeutic substances as anactive ingredient, as long as the substance does not inhibit theantitumoral effect of the active ingredient of the present invention,e.g., any of the peptides, polynucleotides, exosomes, APCs, CTLs of thepresent invention. For example, formulations can includeanti-inflammatory substances, pain killers, chemotherapeutics, and thelike. In addition to including other therapeutic substances in themedicament itself, the medicaments of the present invention can also beadministered sequentially or concurrently with the one or more otherpharmacologic compositions. The amounts of medicament and pharmacologiccomposition depend, for example, on what type of pharmacologiccomposition(s) is/are used, the disease being treated, and thescheduling and routes of administration.

It should be understood that in addition to the ingredients particularlymentioned herein, the pharmaceutical compositions or agent of thepresent invention can include other substances conventional in the arthaving regard to the type of formulation in question.

In one embodiment of the present invention, the pharmaceuticalcompositions or agents of the present invention can be packaged inarticles of manufacture and kits containing materials useful fortreating the pathological conditions of the disease to be treated, e.g.,cancer. The article of manufacture can include a container of any of thepresent pharmaceutical compositions or agents with a label. Suitablecontainers include bottles, vials, and test tubes. The containers can beformed from a variety of materials, such as glass or plastic. The labelon the container should indicate the composition or agent is used fortreating or prevention of one or more conditions of the disease. Thelabel can also indicate directions for administration and so on.

In addition to the container described above, a kit including apharmaceutical composition or agent of the present invention canoptionally further include a second container housing apharmaceutically-acceptable diluent. It can further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The pharmaceutical compositions or agents of the present invention can,if desired, be presented in a pack or dispenser device which can containone or more unit dosage forms containing the active ingredient. The packcan, for example, include metal or plastic foil, such as a blister pack.The pack or dispenser device can be accompanied by instructions foradministration.

(1) Pharmaceutical Compositions Containing the Peptides as ActiveIngredients

The peptide of the present invention can be administered directly as apharmaceutical composition or agent, or if necessary, may be formulatedby conventional formulation methods. In the latter case, in addition tothe peptides of the present invention, carriers, excipients, and suchthat are ordinarily used for drugs can be included as appropriatewithout particular limitations. Examples of such carriers are sterilizedwater, physiological saline, phosphate buffer, culture fluid and such.Furthermore, the pharmaceutical compositions or agents of the presentinvention can contain as necessary, stabilizers, suspensions,preservatives, surfactants and such. The pharmaceutical compositions oragents of the present invention can be used for anti-cancer purposes.

The peptides of the present invention can be prepared in combination,which includes two or more of peptides of the present invention, toinduce CTLs in vivo. The peptides can be in a cocktail or can beconjugated to each other using standard techniques. For example, thepeptides can be chemically linked or expressed as a single fusionpolypeptide. The peptides in the combination can be the same ordifferent. By administering the peptides of the present invention, thepeptides are presented in high density by the HLA antigens on APCs, andthen CTLs that specifically react toward the complex formed between thedisplayed peptide and the HLA antigen are induced. Alternatively, APCs(e.g., DCs) may be removed from a subject and then stimulated by thepeptides of the present invention to obtain APCs that present any of thepeptides of the present invention on their cell surface. These APCs canbe re-administered to the subject to induce CTLs in the subject, and asa result, aggressiveness towards the tumor-associated endothelium can beincreased.

The pharmaceutical compositions or agents for the treatment and/orprevention of cancer that include any of peptides of the presentinvention as active ingredients can also include an adjuvant so thatcellular immunity will be established effectively. Alternatively, thepharmaceutical compositions or agents of the present invention can beadministered with other active ingredients, or can be administered byformulation into granules. An adjuvant refers to any compound, substanceor composition that enhances the immune response against a protein whenadministered together (or successively) with the protein havingimmunological activity. Adjuvants contemplated herein include thosedescribed in the literature (Johnson A G, Clin Microbiol Rev 1994, 7:277-89). Examples of suitable adjuvants include, but are not limited to,aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonellatoxin, IFA (Incomplete Freund's adjuvant), CFA (Complete Freund'sadjuvant), ISCOMatrix, GM-CSF, CpG, O/W emulsion and the like.

Furthermore, liposome formulations, granular formulations in which thepeptide is bound to few-micrometers diameter beads, and formulations inwhich a lipid is bound to the peptide may be conveniently used.

In another embodiment, the peptides of the present invention may also beadministered in the form of a pharmaceutically acceptable salt. Examplesof preferred salts include, but are not limited to, salts with an alkalimetal, salts with a metal, salts with an organic base, salts with anamine, salts with an organic acid (acetic acid, formic acid, propionicacid, fumaric acid, maleic acid, succinic acid, tartaric acid, citricacid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and soon) and salts with an inorganic acid (hydrochloric acid, phosphoricacid, hydrobromic acid , sulfuric acid, nitric acid, and so on). As usedherein, the phrase “pharmaceutically acceptable salt” refers to thosesalts which retain the biological effectiveness and properties of thecompound and which are obtained by reaction with inorganic or organicacids or bases such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicylic acid and the like.

In some embodiments, the pharmaceutical compositions or agents of thepresent invention may further include a component that primes CTLs.Lipids have been identified as substances capable of priming CTLs invivo against viral antigens. For example, palmitic acid residues can beattached to the epsilon- and alpha-amino groups of a lysine residue andthen linked to a peptide of the present invention. The lipidated peptidecan then be administered either directly in a micelle or particle,incorporated into a liposome, or emulsified in an adjuvant. As otherexamples of lipids, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used toprime CTLs when covalently attached to an appropriate peptide (see,e.g., Deres et al., Nature 1989, 342: 561-4).

Examples of suitable methods of administration, include, but are notnecessarily limited to, oral, and intradermal, subcutaneous,intramuscular, intraosseous, peritoneal and intravenous injection, orsuch, and systemic administration or local administration to thevicinity of the targeted sites. The administration can be performed bysingle administration or boosted by multiple administrations. Apharmaceutically or therapeutically effective amount of the peptide ofthe present invention can be administered to a subject in need oftreatment of cancer expressing UBE2T. Alternatively, an amount of thepeptide of the present invention sufficient to enhance or stimulateimmunological response mediated with CTLs, and/or to induce CTLs againstcancer or tumor expressing UBE2T can be administered to a subjectcarrying a cancer expressing UBE2T. The dose of the peptides of thepresent invention can be adjusted appropriately according to the diseaseto be treated, age of the patient, weight, method of administration, andsuch, and is ordinarily 0.001 mg to 1000 mg, for example, 0.01 mg to 100mg, for example, 0.1 mg to 30 mg, for example, 0.1 mg to 10 mg, forexample, 0.5 mg to 5mg, and can be administered once in a few days to afew months, for example, once a week. One skilled in the art canappropriately select a suitable dose.

(2) Pharmaceutical Compositions Containing Polynucleotides as ActiveIngredients

The pharmaceutical compositions or agents of the present invention canalso contain nucleic acids encoding the peptide(s) of the presentinvention in an expressible form. Herein, the phrase “in an expressibleform” means that the polynucleotide, when introduced into a cell, willbe expressed in vivo as a polypeptide that induces anti-tumor immunity.In an exemplified embodiment, the nucleic acid sequence of thepolynucleotide of interest includes regulatory elements necessary forexpression of the polynucleotide. The polynucleotide(s) can be equippedso to achieve stable insertion into the genome of the target cell (see,e.g., Thomas K R & Capecchi M R, Cell 1987, 51: 503-12 for a descriptionof homologous recombination cassette vectors). See, e.g., Wolff et al.,Science 1990, 247: 1465-8; U.S. Pat. Nos. 5,580,859; 5,589,466;5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples ofDNA-based delivery technologies include “naked DNA”, facilitated(bupivacaine, polymers, peptide-mediated) delivery, cationic lipidcomplexes, and particle-mediated (“gene gun”) or pressure-mediateddelivery (see, e.g., U.S. Pat. No. 5,922,687).

The peptides of the present invention can also be expressed by viral orbacterial vectors. Examples of expression vectors include attenuatedviral hosts, such as vaccinia or fowlpox. This approach involves the useof vaccinia virus, e.g., as a vector to express nucleotide sequencesthat encode the peptide. Upon introduction into a host, the recombinantvaccinia virus expresses the immunogenic peptide, and thereby elicits animmune response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848. Anothervector is BCG (Bacille Calmette Guerin). BCG vectors are described inStover et al., Nature 1991, 351: 456-60. A wide variety of other vectorsuseful for therapeutic administration or immunization e.g., adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71;Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo2000, 14: 571-85.

Delivery of a polynucleotide into a patient can be either direct, inwhich case the patient is directly exposed to a polynucleotide-carryingvector, or indirect, in which case, cells are first transformed with thepolynucleotide of interest in vitro, then the cells are transplantedinto the patient. Theses two approaches are known, respectively, as invivo and ex vivo gene therapies.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; MulliganRC, Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993,62: 191-217; Trends in Biotechnology 1993, 11(5): 155-215). Methodscommonly known in the art of recombinant DNA technology that areapplicable to the present invention are described by Ausubel et al. inCurrent Protocols in Molecular Biology (John Wiley & Sons, NY, 1993);and Krieger in Gene Transfer and Expression, A Laboratory Manual(Stockton Press, NY, 1990).

Administration can be performed by oral, or intradermal, subcutaneous,intravenous, intramuscular, intraosseous, or peritoneal injection, orsuch, and systemic administration or local administration to thevicinity of the targeted sites finds use. The administration can beperformed by single administration or boosted by multipleadministrations. A pharmaceutically or therapeutically effective amountof the polynucleotide can be administered to a subject in need oftreatment of cancer expressing UBE2T. Alternatively, an amount of thepolynucleotide of the present invention sufficient to enhance orstimulate immunological response mediated with CTLs, and/or to induceCTLs against cancer or tumor expressing UBE2T can be administered to asubject carrying a cancer expressing UBE2T. The dose of thepolynucleotide in the suitable carrier or cells transformed with thepolynucleotide encoding the peptides of the present invention can beadjusted appropriately according to the disease to be treated, age ofthe patient, weight, method of administration, and such, and isordinarily 0.001 mg to 1000 mg, for example, 0.01 mg to 100 mg, forexample, 0.1 mg to 30 mg, for example, 0.1 mg to 10 mg, for example, 0.5mg to 5 mg, and can be administered once every a few days to once everyfew months, for example, once a week. One skilled in the art canappropriately select the suitable dose.

X. Methods of Using the Peptides, Polynucleotide, Exosomes, APCs andCTLs

The peptides and polynucleotides of the present invention can be usedfor preparing or inducing APCs and CTLs. The exosomes and APCs of thepresent invention can be also used for preparing or inducing CTLs. Thepeptides, polynucleotides, exosomes and APCs can be used in combinationwith any other compounds as long as the additional compounds do notinhibit their CTL inducibility. Thus, any of the aforementionedpharmaceutical compositions or agents of the present invention can beused for preparing or inducing CTLs. In addition thereto, thoseincluding the peptides or polynucleotides can be also used for preparingor inducing APCs as explained below.

(1) Methods of Inducing Antigen-Presenting Cells (APCs)

The present invention provides methods of inducing APCs with CTLinducibility using the peptides or polynucleotides of the presentinvention.

The methods of the present invention include the step of contacting APCswith the peptides of the present invention in vitro, ex vivo or in vivo.For example, the method of contacting an APC with the peptide ex vivocan include steps of:

a: collecting APCs from a subject, and

b: contacting the APCs of step a with the peptide of the presentinvention.

The APCs are not limited to a particular kind of cells and include DCs,Langerhans cells, macrophages, B cells, and activated T cells, which areknown to present proteinaceous antigens on their cell surface so as tobe recognized by lymphocytes. Preferably, DCs can be used since theyhave the strongest CTL inducibility among APCs. Any one of peptides ofthe present invention can be used by itself or in combination with oneor more of other peptides of the present invention and/or one or more ofCTL-inducible peptides derived from TAAs other than UBE2T.

On the other hand, when the peptides of the present invention areadministered to a subject, APCs are contacted with the peptides in vivo,and consequently, APCs with CTL inducibility are induced in the body ofthe subject. Thus, the method of the present invention may includeadministering the peptide of the present invention to a subject toinduce an APC with CTL inducibility in the body of the subject.Similarly, when the polynucleotide of the present invention isadministered to a subject in an expressible form, the peptide of thepresent invention is expressed and contacted with APCs in vivo, andconsequently, APCs with CTL inducibility are induced in the body of thesubject. Thus, the present invention may also include administering thepolynucleotide of the present invention to a subject to induce an APCwith CTL inducibility in the body of the subject. The phrase“expressible form” is described above in section “IX. PharmaceuticalAgents or Compositions (2) Pharmaceutical Agents or CompositionsContaining Polynucleotides as Active Ingredients”.

Furthermore, the method of the present invention may include introducingthe polynucleotide of the present invention into an APC to induce an APCwith CTL inducibility. For example, the method can include steps of:

a: collecting APCs from a subject, and

b: introducing a polynucleotide encoding the peptide of the presentinvention into the APC collected in step a.

Step b can be performed as described above in section “VI.Antigen-Presenting Cells”.

Alternatively, the present invention provides a method for preparing anantigen-presenting cell (APC) which can specifically induce CTL activityagainst UBE2T, wherein the method can include one of the followingsteps:

(a) contacting an APC with a peptide of the present invention in vitro,ex vivo or in vivo; and

(b) introducing a polynucleotide encoding a peptide of the presentinvention into an APC.

Alternatively, the present invention provides methods for inducing anAPC having CTL inducibility, wherein the methods include the stepselected from among:

(a) contacting an APC with the peptide of the present invention; and

(b) introducing the polynucleotide encoding the peptide of the presentinvention into an APC.

In a preferred embodiment, the present invention provides the method ofinducing or preparing an APC having CTL inducibility, such methodincluding one of the following steps:

(a) contacting an APC expressing HLA-A24 with a peptide having an aminoacid sequence selected from among SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13,15, 17, 19, 20, 21, 22, 23, 24, 25 and 27 or modified peptide thereof invitro, ex vivo or in vivo; and

(b) introducing a polynucleotide encoding a peptide having an amino acidsequence selected from among SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17,19, 20, 21, 22, 23, 24, 25 and 27 or modified peptide thereof into anAPC expressing HLA-A24.

APCs induced by the above method present such peptides via HLA-A24 ontheir surface, and can induce CTLs having specific cytotoxic activityagainst cells expressing HLA-A24 and UBE2T.

In another embodiment, the present invention provides the method ofinducing or preparing an APC having CTL inducibility, such methodincluding one of the following steps:

(a) contacting an APC expressing HLA-A2 with a peptide having an aminoacid sequence selected from among SEQ ID NOs: 29, 30, 32, 36, 38, 41,48, 49, 51, 52, 53, 55, 56 and 58 or modified peptide thereof in vitro,ex vivo or in vivo; and

(b) introducing a polynucleotide encoding a peptide having an amino acidsequence selected from among SEQ ID NOs: 29, 30, 32, 36, 38, 41, 48, 49,51, 52, 53, 55, 56 and 58 or modified peptide thereof into an APCexpressing HLA-A2.

APCs induced by the above method present such peptides via HLA-A2 ontheir surface, and can induce CTLs having specific cytotoxic activityagainst cells expressing HLA-A2 and UBE2T.

The methods of the present invention can be carried out in vitro, exvivo or in vivo. Preferably, the methods of the present invention can becarried out in vitro or ex vivo. APCs used for induction of APCs havingCTL inducibility can be preferably APCs expressing HLA-A24 or HLA-A2antigen. Such APCs can be prepared by the methods well-known in the artsfrom peripheral blood mononuclear cells (PBMCs) obtained from a subjectwhose HLA antigen is HLA-A24 or HLA-A2. The APCs induced by the methodof the present invention can be APCs that present a complex of thepeptide of the present invention and HLA antigen (HLA-A24 or HLA-A2antigen) in its surface. When APCs induced by the method of the presentinvention are administered to a subject in order to induce immuneresponses against cancer in the subject, the subject is preferably thesame one from whom APCs are derived. However, the subject may be adifferent one from the APC donor as long as the subject has the same HLAtype with the APC donor.

In another embodiment, the present invention provides agents orcompositions for use in inducing an APC having CTL inducibility, andsuch agents or compositions include one or more peptides orpolynucleotides of the present invention.

In another embodiment, the present invention provides the use of thepeptide of the present invention or the polynucleotide encoding thepeptide in the manufacture of an agent or composition formulated forinducing APCs.

Alternatively, the present invention further provides the peptide of thepresent invention or the polypeptide encoding the peptide for use ininducing an APC having CTL inducibility.

(2) Method of Inducing CTLs

The present invention also provides methods for inducing CTLs using thepeptides, polynucleotides, or exosomes or APCs of the present invention.

The present invention also provides methods for inducing CTLs using apolynucleotide encoding both of TCR subunits or polynucleotides encodingeach of TCR subunits, wherein the TCR formed by such subunits canrecognize (bind to) a complex of the peptide of the present inventionand an HLA antigen on a cell surface. Preferably, the methods forinducing CTLs may include at least one step selected from among:

a: contacting a CD8 positive T cell with an antigen-presenting cell thatpresents on its surface a complex of an HLA antigen and a peptide of thepreset invention

b: contacting a CD8 positive T cell with an exosome that presents on itssurface a complex of an HLA antigen and a peptide of the presetinvention; and

c: introducing a polynucleotide encoding both of TCR subunits orpolynucleotides encoding each of TCR subunits into a CD8 positive Tcell, wherein the TCR formed by such subunits can recognize (bind to) acomplex of a peptide of the present invention and an HLA antigen on acell surface.

When the peptides, polynucleotides, APCs, or exosomes of the presentinvention are administered to a subject, CTLs are induced in the body ofthe subject, and the strength of immune responses targeting cancer cellsexpressing UBE2T is enhanced. Thus, the methods of the present inventioncan include the step of administering the peptides, polynucleotides,APCs or exosomes of the present invention to a subject.

Alternatively, CTLs can be also induced by using them ex vivo or invitro, and after inducing CTLs, the activated CTLs can be returned tothe subject. For example, the method can include steps of:

a: collecting APCs from subject,

b: contacting the APCs of step a, with the peptide of the presentinvention, and

c: co-culturing the APCs of step b with CD8 positive T cells.

The APC to be co-cultured with the CD8 positive T cell in above step ccan also be prepared by transferring a polynucleotide of the presentinvention into an APC as described above in section “VI.Antigen-Presenting Cells”, although the present invention is not limitedthereto and thus encompasses any APCs that effectively present on itssurface a complex of an HLA antigen and a peptide of the presentinvention.

One may optionally utilize exosomes that present on its surface acomplex of an HLA antigen and the peptide of the present inventioninstead of the aforementioned APCs. Namely, the present invention canincludes the step of co-culturing exosomes presenting on its surface acomplex of an HLA antigen and the peptide of the present invention andCD8 positive T cells. Such exosomes can be prepared by the methodsdescribed above in section “V. Exosomes”. Suitable APCs and exosomes forthe method of the present invention present a complex of the peptide ofthe present invention and HLA-A24 or HLA-A2 on its surface.

For example, an APC or exosome that present a complex of an HLA-A24 anda peptide having an amino acid sequence selected from among SEQ ID NOs:1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25 and 27 (ormodified peptide thereof) on its surface can be preferably utilize forinducing a CTL having specific cytotoxic activity against a cellexpressing HLA-A24 and UBE2T. Likewise, an APC or exosome that present acomplex of an HLA-A2 and a peptide having an amino acid sequenceselected from among SEQ ID NOs: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52,53, 55, 56 and 58 (or modified peptide thereof) on its surface can bepreferably utilize for inducing a CTL having specific cytotoxic activityagainst a cell expressing HLA-A2 and UBE2T.

Furthermore, the CTL can be induced by introducing a polynucleotideencoding both of the TCR subunits or polynucleotides encoding each ofthe TCR subunits into CD8 positive T cell, wherein the TCR formed bysuch subunits can bind to a complex of the peptide of the presentinvention and an HLA antigen on a cell surface. Such transduction can beperformed as described above in section “VIII. T Cell Receptor (TCR)”.

The methods of the present invention can be carried out in vitro, exvivo or in vivo. Preferably, the methods of the present invention can becarried out in vitro or ex vivo. CD8 positive T cells used for inductionof CTLs can be prepared by well-known methods in the art from PBMCsobtained from a subject. In preferred embodiments, the donor for CD8positive T cells can be a subject whose HLA antigen is HLA-A24 orHLA-A2. The CTLs induced by the methods of the present invention can beCTLs that can recognize cells presenting a complex of the peptide of thepresent invention and an HLA antigen on its surface. Such CTLs can showspecific cytotoxic activity against cells that present the peptide ofthe present invention on its surface, and therefore, can show specificcytotoxic activity against cells expressing UBE2T (e.g., cancer cells).When CTLs induced by the method of the present invention areadministered to a subject in order to induce immune responses againstcancer in the subject, the subject is preferably the same one from whomCD8 positive T cells are derived. However, the subject may be adifferent one from the CD8 positive T cell donor as long as the subjecthas the same HLA type with the CD8 positive T cell donor.

In addition, the present invention provides a method or process formanufacturing a pharmaceutical composition or agent for inducing a CTL,wherein the method or process includes the step of admixing orformulating the peptide of the present invention with a pharmaceuticallyacceptable carrier.

In another embodiment, the present invention provides an agent orcomposition for inducing a CTL, wherein the agent or compositioncomprises one or more peptide(s), one or more polynucleotide(s), one ormore APCs, and/or one or more exosomes of the present invention.

In another embodiment, the present invention provides the use of thepeptide, polynucleotide, APC or exosome of the present invention in themanufacture of an agent or composition formulated for inducing a CTL.

Alternatively, the present invention further provides the peptide,polynucleotide, APC or exosome of the present invention for use ininducing a CTL.

XI. Methods of Inducing Immune Response

Moreover, the present invention provides methods of inducing immuneresponses against diseases related to UBE2T. Suitable diseases includecancer, examples of which include, but are not limited to, bladdercancer, breast cancer, cervical cancer, cholangiocellular carcinoma,CML, colorectal cancer, esophageal cancer, gastric cancer, diffuse-typegastric cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer,pancreatic cancer, prostate cancer, SCLC, soft tissue tumor andtesticular tumor.

The methods of the present invention may include the step ofadministering an agent or composition containing any of the peptides ofthe present invention or polynucleotides encoding them. The inventivemethods also contemplate the administration of exosomes or APCspresenting any of the peptides of the present invention. For details,see the item of “IX. Pharmaceutical Agents or Compositions”,particularly the part describing the use of the pharmaceuticalcompositions of the present invention as vaccines. In addition, theexosomes and APCs that can be employed for the present methods forinducing immune response are described in detail under the items of “V.Exosomes”, “VI. Antigen-Presenting Cells (APCs)”, and (1) and (2) of “X.Methods Using the Peptides, Exosomes, APCs and CTLs”, supra.

The present invention also provides a method or process formanufacturing a pharmaceutical composition or agent for inducing immuneresponse against cancer, wherein the method may include the step ofadmixing or formulating the peptide of the present invention with apharmaceutically acceptable carrier.

Alternatively, the method of the present invention may include the stepof administrating a vaccine or a pharmaceutical composition or agent ofthe present invention that contains:

(a) a peptide of the present invention;

(b) a polynucleotide encoding the peptide of the present invention in anexpressible form;

(c) an APC presenting the peptide of the present invention on itssurface;

(d) an exosome presenting the peptide of the present invention on itssurface; or

(e) a CTL of the present invention.

In the context of the present invention, a cancer over-expressing UBE2Tcan be treated with these active ingredients. Examples of such cancerinclude, but are not limited to, bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer, esophagealcancer, gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor. Accordingly, prior to theadministration of the vaccines or pharmaceutical compositions or agentincluding any of aforementioned active ingredients, it is preferable toconfirm whether the expression level of UBE2T in cancerous cells ortissues collected from the subject to be treated is elevated as comparedwith normal cells or tissues collected from the same subject. Thus, inone embodiment, the present invention provides a method for treatingcancer (over)expressing UBE2T in a patient in need thereof, such methodincluding the steps of:

i) determining the expression level of UBE2T in a biological sampleobtained from a subject with the cancer to be treated;

ii) comparing the expression level of UBE2T with normal control; and

iii) administrating at least one component selected from among (a) to(e) described above to a subject with cancer over-expressing UBE2Tcompared with normal control.

Alternatively, the present invention provides a vaccine orpharmaceutical composition including at least one component selectedfrom among (a) to (e) described above, to be administered to a subjecthaving cancer over-expressing UBE2T. In other words, the presentinvention further provides a method for identifying a subject to betreated with the peptide of the present invention, such method includingthe step of determining an expression level of UBE2T in asubject-derived biological sample, wherein an increase of the expressionlevel as compared to a normal control level of the gene indicates thatthe subject may have cancer which may be treated with the peptide of thepresent invention.

Further, in preferred embodiments, the HLA type of a subject may beidentified before administering the peptides of the present invention.For example, peptides having the amino acid sequence of SEQ ID NO: 1, 2,4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, or 27 arepreferably administered to a subject identified as having HLA-A24.Alternatively, peptides having the amino acid sequence of SEQ ID NO: 29,30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58 are preferablyadministered to a subject identified as having HLA-A2.

Any subject-derived cell or tissue can be used for the determination ofthe expression level of UBE2T as long as it can include thetranscription or translation product of UBE2T. Examples of suitablesamples include, but are not limited to, bodily tissues and fluids, suchas blood, sputum and urine. Preferably, the subject-derived cell ortissue sample contains a cell population including an epithelial cell,more preferably a cancerous epithelial cell or an epithelial cellderived from cancerous tissue. Further, if necessary, the cell may bepurified from the obtained bodily tissues and fluids, and then used asthe subjected-derived sample.

According to the present invention, the expression level of UBE2T in abiological sample obtained from a subject may be determined. Theexpression level of UBE2T can be determined at the transcription(nucleic acid) product level, using methods known in the art. Forexample, the mRNA of UBE2T may be quantified using probes byhybridization methods (e.g., Northern hybridization). The detection maybe carried out on a chip or an array. The use of an array is preferablefor detecting the expression level of UBE2T. Those skilled in the artcan prepare such probes utilizing the sequence information of UBE2T. Forexample, the cDNA of UBE2T may be used as the probes. If necessary, theprobes may be labeled with a suitable label, such as dyes, fluorescentsubstances and isotopes, and the expression level of UBE2T may bedetected as the intensity of the hybridized labels.

Furthermore, the transcription product of UBE2T may be quantified usingprimers by amplification-based detection methods (e.g., RT-PCR). Suchprimers may be prepared based on the available sequence information ofUBE2T.

Specifically, a probe or primer used for the present method hybridizesunder stringent, moderately stringent, or low stringent conditions tothe mRNA of UBE2T. As used herein, the phrase “stringent (hybridization)conditions” refers to conditions under which a probe or primer willhybridize to its target sequence, but not to other sequences. Stringentconditions are sequence-dependent and will be different under differentcircumstances. Specific hybridization of longer sequences is observed athigher temperatures than shorter sequences. Generally, the temperatureof a stringent condition is selected to be about 5 degree Centigradelower than the thermal melting point (Tm) for a specific sequence at adefined ionic strength and pH. The Tm is the temperature (under adefined ionic strength, pH and nucleic acid concentration) at which 50%of the probes complementary to their target sequence hybridize to thetarget sequence at equilibrium. Since the target sequences are generallypresent at excess, at Tm, 50% of the probes are occupied at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30 degree Centigrade for short probes orprimers (e.g., 10 to 50 nucleotides) and at least about 60 degreeCentigrade for longer probes or primers. Stringent conditions may alsobe achieved with the addition of destabilizing substances, such asformamide.

A probe or primer of the present invention is typically a substantiallypurified oligonucleotide. The oligonucleotide typically includes aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100,50, or 25, consecutive sense strand nucleotide sequence of a nucleicacid including a UBE2T sequence, or an anti-sense strand nucleotidesequence of a nucleic acid including a UBE2T sequence, or of a naturallyoccurring mutant of these sequences. In particular, for example, in apreferred embodiment, an oligonucleotide having 5-50 in length can beused as a primer for amplifying the genes, to be detected. Morepreferably, mRNA or cDNA of a UBE2T gene can be detected witholigonucleotide probe or primer of a specific size, generally 15-30bases in length. The size may range from at least 10 nucleotides, atleast 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides,at least 25 nucleotides, at least 30 nucleotides and the probes andprimers may range in size from 5-10 nucleotides, 10-15 nucleotides,15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides. In preferredembodiments, length of the oligonucleotide probe or primer can beselected from 15-25 nucleotides. Assay procedures, devices, or reagentsfor the detection of gene by using such oligonucleotide probe or primerare well known (e.g. oligonucleotide microarray or PCR). In theseassays, probes or primers can also include tag or linker sequences.Further, probes or primers can be modified with detectable label oraffinity ligand to be captured. Alternatively, in hybridization baseddetection procedures, a polynucleotide having a few hundreds (e.g.,about 100-200) bases to a few kilo (e.g., about 1000-2000) bases inlength can also be used for a probe (e.g., northern blotting assay orcDNA microarray analysis).

Alternatively, the translation product of UBE2T may be detected for theidentification of a subject to be treated by the method of the presentinvention. For example, the quantity of UBE2T protein (SEQ ID NO: 65)may be determined. Examples of methods for determining the quantity ofthe UBE2T protein as the translation product include immunoassay methodsusing an antibody specifically recognizing the UBE2T protein. Theantibody may be monoclonal or polyclonal. Furthermore, any fragment ormodification (e.g., chimeric antibody, scFv, Fab, F(ab′)₂, Fv, etc.) ofthe antibody may be used for the detection, as long as the fragment ormodified antibody retains the binding ability to the UBE2T protein.Methods to prepare these kinds of antibodies are well known in the art,and any method may be employed to prepare such antibodies andequivalents thereof.

As another method to detect the expression level of UBE2T based on itstranslation product, the intensity of staining may be measured viaimmunohistochemical analysis using an antibody against the UBE2Tprotein. Namely, in this measurement, strong staining indicatesincreased presence/level of the UBE2T protein and, at the same time,high expression level of UBE2T.

The expression level of the UBE2T gene in a subject-derived sample canbe determined to be increased if the expression level increases from thecontrol level (e.g., the expression level in normal cells) of the UBE2Tby, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold,more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than10.0 fold, or more.

The control level may be determined at the same time as the cancer cellsby using a sample(s) previously collected and stored from a healthysubject/subjects. In addition, normal cells obtained from non-cancerousregions of an organ that has the cancer to be treated may be used asnormal control. Alternatively, the control level may be determined by astatistical method based on the results obtained by analyzing previouslydetermined expression level(s) of UBE2T in samples from subjects whosedisease states are known. Furthermore, the control level can be derivedfrom a database of expression patterns from previously tested cells.Moreover, according to an aspect of the present invention, theexpression level of UBE2T in a biological sample may be compared tomultiple control levels, which are determined from multiple referencesamples. It is preferred to use a control level determined from areference sample derived from a tissue type similar to that of thesubject-derived biological sample. Moreover, it is preferred to use thestandard value of the expression levels of UBE2T gene in a populationwith a known disease state. The standard value may be obtained by anymethod known in the art. For example, a range of mean +/−2 S.D. or mean+/−3 S.D. may be used as the standard value.

Difference between a sample expression level and a control level can benormalized to the expression level of control nucleic acids, e.g.,housekeeping genes, whose expression levels are known not to differdepending on the cancerous or non-cancerous state of the cell. Exemplarycontrol genes include, but are not limited to, beta-actin,glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.

When the expression level of UBE2T is increased as compared to thenormal control level, the subject may be identified as a subject withcancer to be treated by administration of the pharmaceutical compositionor agent of the present invention.

The present invention also provides a method of selecting a subject forcancer treatment using aforementioned pharmaceutical compositions oragents of the present invention, such method including the steps of:

a) determining the expression level of UBE2T in biological sample(s)obtained from a subject with cancer;

b) comparing the expression level of UBE2T determined in step a) with anormal control level; and

c) selecting the subject for cancer treatment by the pharmaceuticalcompositions or agents of the present invention, if the expression levelof UBE2T is increased as compared to the normal control level.

In some embodiments, such a method may further comprise the step ofidentifying, after or before the steps a)-c) defined above, a subjecthaving an HLA selected from the group consisting of HLA-A24 and HLA-A2.Cancer therapy according to the present invention is preferable for asubject that suffers from cancer overexpressing UBE2T and has HLA-A24 orHLA-A2. Methods for HLA typing are well known in the art. For example,PCR-based methods for typing HLA alleles are well known. Antibodiesspecific for each HLA molecule are also appropriate tools foridentifying HLA types of a subject.

In one embodiment, the present invention further provides a diagnostickit including one or more peptide of the present invention.

Cancer can be diagnosed by detecting antibodies against the peptide ofthe present invention in a subject-derived sample (e.g., blood) usingthe peptide of the present invention.

The subject is suspected to be suffering from cancer, if asubject-derived sample (e.g., blood sample) contains antibodies againstthe peptide of the present invention and the quantity of the antibodiesis determined to be more than the cut off value as compared to controllevel.

In another embodiment, a diagnostic kit of the present invention mayinclude the peptide of the present invention and an HLA molecule bindingthereto. The method for detecting antigen specific CTLs using antigenicpeptides and HLA molecules has already been established (for example,Altman JD et al., Science. 1996, 274(5284): 94-6). Thus, the complex ofthe peptide of the present invention and the HLA molecule can be appliedto the detection method to detect tumor antigen specific CTLs, therebyenabling earlier detection, recurrence and/or metastasis of cancer.Further, it can be employed for the selection of subjects applicablewith the pharmaceutical composition or agent including the peptide ofthe present invention as an active ingredient, or the assessment of thetreatment effect of the pharmaceutical composition or agent.

Particularly, according to the known method (see, for example, Altman JD et al., Science. 1996, 274(5284): 94-6), the oligomer complex, such astetramer, of the radiolabeled HLA molecule and the peptide of thepresent invention can be prepared. With using the complex, the diagnosiscan be done, for example, by quantifying the antigen-peptide specificCTLs in the peripheral blood lymphocytes derived from a subjectsuspected to be suffering from cancer.

The present invention further provides methods and diagnostic agents forevaluating immunological response of subject by using the peptide of thepresent invention. In one embodiment of the invention, the peptides ofthe present invention are used as reagents for evaluating or predictingan immune response of a subject. The immune response to be evaluated isinduced by contacting an immunogen (i.e., the peptide of the presentinvention) with immunocompetent cells in vitro or in vivo. In preferredembodiments, the immunocompetent cells for evaluating an immunologicalresponse, may be selected from among peripheral blood, peripheral bloodlymphocyte (PBL), and peripheral blood mononuclear cell (PBMC). Assaysystems that are used for such an analysis include relatively recenttechnical developments such as tetramer staining assays, staining forintracellular lymphokine and interferon release assays, or ELISPOTassays. In a preferred embodiment, immunocompetent cells to be contactedwith the peptide reagent may be antigen presenting cells includingdendritic cells.

For example, the peptides of the present invention may be used intetramer staining assays to assess peripheral blood mononuclear cellsfor the presence of antigen-specific CTLs following exposure to a tumorcell antigen or an immunogen. The HLA tetrameric complex may be used todirectly visualize antigen specific CTLs (see, e.g., Ogg et al., Science279: 2103-2106, 1998; and Altman et al, Science 174: 94-96, 1996) anddetermine the frequency of the antigen-specific CTL population in asample of peripheral blood mononuclear cells. A tetramer reagent using apeptide of the invention may be generated as described below.

A peptide that binds to an HLA molecule is refolded in the presence ofthe corresponding HLA heavy chain and beta 2-microglobulin to generate atrimolecular complex. In the complex, carboxyl terminal of the heavychain is biotinylated at a site that was previously engineered into theprotein. Then, streptavidin is added to the complex to form tetramercomposed of the trimolecular complex and streptavidin. By means offluorescently labeled streptavidin, the tetramer can be used to stainantigen-specific cells. The cells can then be identified, for example,by flow cytometry. Such an analysis may be used for diagnostic orprognostic purposes. Cells identified by the procedure can also be usedfor therapeutic purposes.

The peptides of the present invention may be also used to makeantibodies, using techniques well known in the art (see, e.g., CURRENTPROTOCOLS IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A LaboratoryManual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989),which may be useful as reagents to diagnose or monitor cancer. Suchantibodies may include those that recognize a peptide in the context ofan HLA molecule, i. e., antibodies that bind to a peptide-MHC complex.

The peptides and compositions of the present invention have a number ofadditional uses, some of which are described herein. For instance, thepresent invention provides a method for diagnosing or detecting adisorder characterized by expression of a UBE2T polypeptide.

For example, the diagnosis can be done, by a method which allows directquantification of antigen-specific T cells by staining withFluorescein-labelled HLA multimeric complexes (for example, Altman, J.D. et al., 1996, Science 274: 94; Altman, J. D. et al., 1993, Proc.Natl. Acad. Sci. USA 90: 10330;). Staining for intracellularlymphokines, and interferon-gamma release assays or ELISPOT assays alsohas been provided. Tetramer staining, intracellular lymphokine stainingand ELISPOT assays all appear to be at least 10-fold more sensitive thanmore conventional assays (Murali-Krishna K. et al., 1998, Immunity 8:177; Lalvani A. et al., 1997, J. Exp. Med. 186: 859; Dunbar P. R. etal., 1998, Curr. Biol. 8: 413;). Pentamers (e.g., US 2004-209295A),dextramers (e.g., WO 02/072631), and streptamers (e.g., Nature medicine6. 631-637 (2002)) may also be used.

For instance, in some embodiments, the present invention provides amethod for diagnosing or evaluating an immunological response of asubject administered at least one of UBE2T peptides of the presentinvention, the method including the steps of:

(a) contacting an immunogen with immunocompetent cells under thecondition suitable for induction of CTL specific to the immunogen;

(b) detecting or determining induction level of the CTL induced in step(a); and

(c) correlating the immunological response of the subject with the CTLinduction level.

In the present invention, the immunogen is at least one of UBE2Tpeptides having the amino acid sequences of SEQ ID NO: 1, 2, 4, 6, 11,12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38, 41,48, 49, 51, 52, 53, 55, 56 or 58 , and peptides having in which suchamino acid sequences have been modified with 1, 2 or more amino acidsubstitution(s). In the meantime, conditions suitable of induction ofimmunogen specific CTLs are well known in the art. For example,immunocompetent cells may be cultured in vitro under the presence ofimmunogen(s) to induce immunogen specific CTLs. In order to induceimmunogen specific CTLs, any stimulating factors may be added to thecell culture. For example, IL-2 is preferable stimulating factors forthe CTL induction.

In some embodiments, the step of monitoring or evaluating immunologicalresponse of a subject to be treated with peptide cancer therapy may beperformed before, during and/or after the treatment. In general, duringa protocol of cancer therapy, immunogenic peptides are administeredrepeatedly to a subject to be treated. For example, immunogenic peptidesmay be administered every week for 3-10 weeks. Accordingly, theimmunological response of the subject can be evaluated or monitoredduring the cancer therapy protocol. Alternatively, the step ofevaluation or monitoring of immunological response to the cancer therapymay at the completion of the therapy protocol.

According to the present invention, enhanced induction of immunogenspecific CTLs as compared with a control indicates that the subject tobe evaluated or diagnosed immunologically responded to the immunogen(s)that has/have been administered. Suitable controls for evaluating theimmunological response may include, for example, a CTL induction levelwhen the immunocompetent cells are contacted with no peptide, or controlpeptide(s) having amino acid sequences other than any UBE2T peptides(e.g., random amino acid sequence).

XII. Antibodies

The present invention further provides antibodies that bind to peptidesof the present invention. Preferred antibodies specifically bind topeptides of the present invention and will not bind (or will bindweakly) to those other than the peptides of the present invention.

Antibodies against the peptides of the present invention can find use incancer diagnostic and prognostic assays. Similarly, such antibodies canfind use in the treatment, diagnosis, and/or prognosis of cancers, tothe extent UBE2T is also expressed or over-expressed in cancer.Moreover, intracellularly expressed antibodies (e.g., single chainantibodies) may therapeutically find use in treating cancers in whichthe expression of UBE2T is involved, example of which include, but arenot limited to, bladder cancer, breast cancer, cervical cancer,cholangiocellular carcinoma, CML, colorectal cancer, esophageal cancer,gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor.

The present invention also provides various immunological assays for thedetection and/or quantification of the UBE2T protein (SEQ TD NO: 65) orfragments thereof, including peptides consisting of the amino acidsequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 6,11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38,41, 48, 49, 51, 52, 53, 55, 56 and 58. In the context of the presentinvention, antibodies binding to UBE2T polypeptide preferably recognizethe peptide consisting of the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20,21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55,56 and 58. A binding specificity of an antibody can be confirmed withinhibition test. That is, when the binding between an antibody to beanalyzed and full-length of UBE2T polypeptide is inhibited under thepresence of any fragment consisting of the amino acid sequence of SEQ IDNO: 1, 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29,30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58, it is shown thatthis antibody specifically binds to the fragment. In the context of thepresent invention, such immunological assays are performed withinvarious immunological assay formats well known in the art, including butnot limited to, various types of radioimmunoassays, immuno-chromatographtechnique, enzyme-linked immunosorbent assays (ELISA), enzyme-linkedimmunofluorescent assays (ELIFA), and the like.

Related immunological but non-antibody assays may include T cellimmunogenicity assays (inhibitory or stimulatory) as well as MHC bindingassays. In addition, the present invention contemplates immunologicalimaging methods capable of detecting cancers expressing UBE2T, exampleof which include, but are not limited to, radioscintigraphic imagingmethods using labeled antibodies of the present invention. Such assaysfind clinical use in the detection, monitoring, and prognosis of UBE2Texpressing cancers, examples of which include, but are not limited to,bladder cancer, breast cancer, cervical cancer, cholangiocellularcarcinoma, CML, colorectal cancer, esophageal cancer, gastric cancer,diffuse-type gastric cancer, NSCLC, lymphoma, osteosarcoma, ovariancancer, pancreatic cancer, prostate cancer, SCLC, soft tissue tumor andtesticular tumor.

The antibody of the present invention can be used in any form, forexample as a monoclonal or polyclonal antibody, and may further includeanti-serum obtained by immunizing an animal such as a rabbit with thepeptide of the present invention, all classes of polyclonal andmonoclonal antibodies, human antibodies and humanized antibodiesproduced by genetic recombination.

The antibody of the present invention can recognize peptides having anamino acid sequence selected from the group consisting of SEQ ID NOs: 1,2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32,36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58. Methods for synthesizingoligopeptide are well known in the arts. After the synthesis, peptidesmay be optionally purified prior to use as immunogen. In the context ofthe present invention, the oligopeptide (e.g., 9 or 10mer) may beconjugated or linked with carriers to enhance the immunogenicity.Keyhole-limpet hemocyanin (KLH) is well known as the carrier. Method forconjugating KLH and peptide are also well known in the arts.

Alternatively, a gene encoding a peptide of the present invention may beinserted into a known expression vector, which is then used to transforma host cell as described herein. The desired peptide may be recoveredfrom the outside or inside of host cells by any standard method, and maysubsequently be used as an antigen. Alternatively, whole cellsexpressing the peptide or their lysates or a chemically synthesizedpeptide may be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha or Primates may beused. Animals of the family Rodentia include, for example, mouse, ratand hamster. Animals of the family Lagomorpha include, for example,rabbit. Animals of the Primate family include, for example, a monkey ofCatarrhini (old world monkey) such as Macaca fascicularis, rhesusmonkey, sacred baboon and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for the immunization of mammals. More specifically,antigens may be diluted and suspended in an appropriate amount ofphosphate buffered saline (PBS), physiological saline, etc. If desired,the antigen suspension may be mixed with an appropriate amount of astandard adjuvant, such as Freund's complete adjuvant, made intoemulsion and then administered to mammalian animals. Preferably, it isfollowed by several administrations of antigen mixed with anappropriately amount of Freund's incomplete adjuvant every 4 to 21 days.An appropriate carrier may also be used for immunization. Afterimmunization as above, serum may be examined by a standard method for anincrease in the amount of desired antibodies.

Polyclonal antibodies against the peptides of the present invention maybe prepared by collecting blood from the immunized mammal examined forthe increase of desired antibodies in the serum, and by separating serumfrom the blood by any conventional method. Polyclonal antibodies mayinclude serum containing the polyclonal antibodies, as well as thefraction containing the polyclonal antibodies may be isolated from theserum. Immunoglobulin G or M can be prepared from a fraction whichrecognizes only the peptide of the present invention using, for example,an affinity column coupled with the peptide of the present invention,and further purifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion may preferably beobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammalians,and more preferably myeloma cells having an acquired property for theselection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre andMilstein, Methods Enzymol 73: 3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all the other cells,with the exception of the desired hybridoma (non-fused cells), to die.Then, the standard limiting dilution may be performed to screen andclone a hybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal isimmunized with an antigen for preparing hybridoma, human lymphocytessuch as those infected by EB virus may be immunized with a peptide,peptide expressing cells or their lysates in vitro. Then, the immunizedlymphocytes are fused with human-derived myeloma cells that are capableof indefinitely dividing, such as U266, to yield a hybridoma producing adesired human antibody that is able to bind to the peptide can beobtained (Unexamined Published Japanese Patent Application No. S63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are extracted. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography or an affinity column to which the peptide of the presentinvention is coupled. The antibody of the present invention can be usednot only for purification and detection of the peptide of the presentinvention, but also as a candidate for agonists and antagonists of thepeptide of the present invention.

Monoclonal antibodies obtained can be also recombinantly prepared usinggenetic engineering techniques (see, for example, Borrebaeck andLarrick, Therapeutic Monoclonal Antibodies, published in the UnitedKingdom by MacMillan Publishers LTD (1990)). For example, a DNA encodingan antibody may be cloned from an immune cell, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, as long as it binds to the peptide ofthe present invention. For instance, the antibody fragment may be Fab,F(ab′)₂, Fv or single chain Fv (scFv), in which Fv fragments from H andL chains are ligated by an appropriate linker (Huston et al., Proc NatlAcad Sci USA 85: 5879-83 (1988)). More specifically, an antibodyfragment may be generated by treating an antibody with an enzyme, suchas papain or pepsin. Alternatively, a gene encoding the antibodyfragment may be constructed, inserted into an expression vector andexpressed in an appropriate host cell (see, for example, Co, et al., JImmunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515(1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al.,Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9:132-7 (1991)).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides forsuch modified antibodies. The modified antibody can be obtained bychemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, between a variable region derived from nonhumanantibody and the constant region derived from human antibody, or as ahumanized antibody, including the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) and theconstant region derived from human antibody. Such antibodies can beprepared according to known technology. Humanization can be performed bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody (see, e.g., Verhoeyen et al., Science239:1534-1536 (1988)). Accordingly, such humanized antibodies arechimeric antibodies, wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

Fully human antibodies including human variable regions in addition tohuman framework and constant regions can also be used. Such antibodiescan be produced using various techniques known in the art. For example,in vitro methods involve use of recombinant libraries of human antibodyfragments displayed on bacteriophage (e.g., Hoogenboom & Winter, J. Mol.Biol. 227:381 (1991). Similarly, human antibodies can be made byintroducing of human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. This approach is described, e.g., in U.S. Pat.Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to the separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by the appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis and isoelectric focusing (Antibodies: A LaboratoryManual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)),but are not limited thereto. A protein A column and protein G column canbe used as the affinity column. Exemplary protein A columns to be usedincludes, for example, Hyper D, POROS and Sepharose F. F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse phase chromatography, adsorption chromatography andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). The chromatographic procedures can becarried out by liquid-phase chromatography, such as HPLC and FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and/orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, a peptide of the present inventionis applied to the plate, and then a sample containing a desiredantibody, such as culture supernatant of antibody producing cells orpurified antibodies, is applied. Then, a secondary antibody thatrecognizes the primary antibody and is labeled with an enzyme, such asalkaline phosphatase, is applied, and the plate is incubated. Next,after washing, an enzyme substrate, such as p-nitrophenyl phosphate, isadded to the plate, and the absorbance is measured to evaluate theantigen binding activity of the sample. BIAcore (Pharmacia) may be usedto evaluate the activity of the antibody of the present invention.

XIII. Vectors and Host Cells

The present invention also provides a vector and host cell into which apolynucleotide encoding the peptide of the present invention isintroduced. A vector of the present invention may be used to keep apolynucleotide, especially a DNA, of the present invention in host cell,to express a peptide of the present invention, or to administer apolynucleotide of the present invention for gene therapy.

When E. coli is a host cell and the vector is amplified and produced ina large amount in E. coli (e.g., JM109, DH5 alpha, HB101 or XL1Blue),the vector should have “ori” to be amplified in E. coli and a markergene for selecting transformed E. coli (e.g., a drug-resistance geneselected by a drug such as ampicillin, tetracycline, kanamycin,chloramphenicol or the like). For example, M13-series vectors,pUC-series vectors, pBR322, pBluescript, pCR-Script, etc., can be used.In addition, pGEM-T, pDIRECT and pT7 can also be used for subcloning andextracting cDNA as well as the vectors described above. When a vector isused to produce the peptide of the present invention, an expressionvector can find use. For example, an expression vector to be expressedin E. coli should have the above characteristics to be amplified in E.coli. When E. coli, such as JM109, DH5 alpha, HB101 or XL 1 Blue, areused as a host cell, the vector should have a promoter, for example,lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7(1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7promoter or the like, that can efficiently express the desired gene inE. coli. In that respect, pGEX-5X-1 (Pharmacia), “QIAexpress system”(Qiagen), pEGFP and pET (in this case, the host is preferably BL21 whichexpresses T7 RNA polymerase), for example, can be used instead of theabove vectors. Additionally, the vector may also contain a signalsequence for peptide secretion. An exemplary signal sequence thatdirects the peptide to be secreted to the periplasm of the E. coli isthe pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)).Means for introducing of the vectors into the target host cells include,for example, the calcium chloride method, and the electroporationmethod.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Mizushima S,etal., Nucleic Acids Res 18(17): 5322 (1990)), pEF, pCDM8), expressionvectors derived from insect cells (for example, “Bac-to-BAC baculovirusexpression system” (GIBCO BRL), pBacPAK8), expression vectors derivedfrom plants (e.g., pMH1, pMH2), expression vectors derived from animalviruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived fromretroviruses (e.g., pZIpneo), expression vector derived from yeast(e.g., “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01) andexpression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50)can be used for producing the peptide of the present invention.

In order to express the vector in animal cells, such as CHO, COS orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al., Nature277: 108 (1979)), the MMLV-LTR promoter, the EF1 alpha promoter(Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMV promoterand the like, and preferably a marker gene for selecting transformants(for example, a drug resistance gene selected by a drug (e.g., neomycin,G418)). Examples of known vectors with these characteristics include,for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Experimental 1 Materials and Methods Cell Lines

TISI, HLA-A*2402-positive B-lymphoblastoid cell line, was purchased fromthe IHWG Cell and Gene Bank (Seattle, Wash.). T2, HLA-A*0201-positiveB-lymphoblastoid cell line, and COS7, African green monkey kidney cellline, was purchased from ATCC.

Candidate Selection of Peptides Derived from UBE2T

9-mer and 10-mer peptides derived from UBE2T that bind to HLA-A*2402 orHLA-A*0201 molecule were predicted using “NetMHC3.2” binding predictionserver (http://www.cbs.dtu.dk/services/NetMHC/) (Buus et al., TissueAntigens. 2003 November, 62(5):378-84; Nielsen et al., Protein Sci. 2003May, 12(5):1007-17, Bioinformatics. 2004 Jun. 12;20(9):1388-97) and“BIMAS” (http://www-bimas.cit.nih.gov/molbio/hla_bind) (Parker et al., JImmunol 1994, 152(1): 163-75, Kuzushima et al., Blood 2001, 98(6):1872-81). These peptides were synthesized by Biosynthesis (Lewisville,Tex.) according to a standard solid phase synthesis method and purifiedby reversed phase high performance liquid chromatography (HPLC). Thepurity (>90%) and the identity of the peptides were determined byanalytical HPLC and mass spectrometry analysis, respectively. Peptideswere dissolved in dimethylsulfoxide at 20 mg/ml and stored at −80degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presentingcells to induce cytotoxic T lymphocyte (CTL) responses against peptidespresented on human leukocyte antigen (HLA). DCs were generated in vitroas described elsewhere (Nakahara S et al., Cancer Res 2003, 63(14):4112-8). Specifically, peripheral blood mononuclear cells isolated froma normal volunteer (HLA-A*2402 or HLA-A*0201 positive) by Ficoll-Plaque(Pharmacia) solution were separated by adherence to a plastic tissueculture dish (Becton Dickinson) so as to enrich them as the monocytefraction. The monocyte-enriched population was cultured in the presenceof 1000 U/ml of granulocyte-macrophage colony-stimulating factor (R&DSystem) and 1000 U/ml of interleukin (IL)-4 (R&D System) in AIM-V Medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS). After7 days of culture, the cytokine-induced DCs were pulsed with 20micro-g/ml of each of the synthesized peptides in the presence of 3micro-g/ml of beta 2-microglobulin for 3 hr at 37 degrees C. in AIM-VMedium. The generated cells appeared to express DC-associated molecules,such as CD80, CD83, CD86 and HLA class II, on their cell surfaces (datanot shown). These peptide-pulsed DCs were then inactivated byX-irradiation (20 Gy) and mixing at a 1:20 ratio with autologous CD8⁺Tcells, obtained by positive selection with CD8 Positive Isolation Kit(Dynal). These cultures were set up in 48-well plates (Corning); eachwell contained 1.5×10⁴ peptide-pulsed DCs, 3×10⁵ CD8⁺T cells and 10ng/ml of IL-7 (R&D System) in 0.5 ml of AIM-V/2% AS medium. Three dayslater, these cultures were supplemented with IL-2 (CHIRON) to a finalconcentration of 20 IU/ml. On day 7 and 14, the T cells were furtherstimulated with the autologous peptide-pulsed DCs. The DCs were preparedeach time by the same way described above. CTLs were tested againstpeptide-pulsed TISI cells or peptide-pulsed TS cells after the 3rd roundof peptide stimulation on day 21 (Tanaka H et al., Br J Cancer 2001,84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida Net al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).

CTL Expansion Procedure

CTLs were expanded in culture using the method similar to the onedescribed by Riddell et al. (Walter E A et al., N Engl J Med 1995333(16): 1038-44; Riddell S R et al., Nat Med 1996, 2(2): 216-23). Atotal of 5×10⁴ CTLs were suspended in 25 ml of AIM-V/5% AS medium with 2kinds of human B-lymphoblastoid cell lines, inactivated by Mitomycin C,in the presence of 40 ng/ml of anti-CD3 monoclonal antibody(Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2were added to the cultures. The cultures were fed with fresh AIM-V/5% ASmedium containing 30 IU/ml of IL-2 on days 5, 8 and 11 (Tanaka H et al.,Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda Tet al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci2005, 96(8): 498-506).

Establishment of CTL Clones

The dilutions were made to have 0.3, 1, and 3 CTLs/well in 96round-bottomed micro titer plate (Nalge Nunc International). CTLs werecultured with 1×10⁴ cells/well of 2 kinds of human B-lymphoblastoid celllines, 30 ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in a total of150 micro-l/well of AIM-V Medium containing 5% AS. 50 micro-1 /well ofIL-2 were added to the medium 10 days later so to reach a finalconcentration of 125 U/ml IL-2. CTL activity was tested on the 14th day,and CTL clones were expanded using the same method as described above(Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al.,Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005,96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, interferon (IFN)-gamma enzyme-linkedimmunospot (ELISPOT) assay and IFN-gamma enzyme-linked immunosorbentassay (ELISA) were performed. Specifically, peptide-pulsed TISI cells orpeptide-pulsed T2 cells (1×10⁴/well) were prepared as stimulator cells.Cultured cells in 48 wells were used as responder cells. IFN-gammaELISPOT assay and IFN-gamma ELISA assay were performed according tomanufacture's procedure.

Establishment of the Cells Forcibly Expressing Either or Both of theTarget Gene and HLA-A24 or HLA-A2

The cDNA encoding an open reading frame of UBE2T, HLA-A*2402 orHLAA*0201 was amplified by PCR. The PCR-amplified product was clonedinto an expression vector. The vectors were transfected into COST cells,which are UBE2T-null, HLA-A*2402-null and HLA-A*0201-null cell line,using lipofectamine 2000 (Invitrogen) according to the manufacturer'srecommended procedures. After 2 days from transfection, the transfectedcells were harvested with versene (Invitrogen) and used as the targetcells (5×10⁴ cells/ well) for CTL activity assay.

Results Enhanced UBE2T Expression in Cancers

The wide gene expression profile data obtained from various cancersusing cDNA-microarray revealed that UBE2T (GenBank Accession No. NM014176 (SEQ ID No: 64)) expression was elevated. UBE2T expression wasvalidly elevated in 24 out of 24 bladder cancers, 44 out of 50 breastcancers, 14 out of 15 cervical cancers, 12 out of 12 cholangiocellularcarcinomas, 9 out of 16 CMLs, 9 out of 9 colorectal cancers, 31 out of47 esophageal cancers, 5 out of 8 gastric cancers, 2 out of 2diffuse-type gastric cancers, 23 out of 27 NSCLCs, 3 out of 3 lymphomas,9 out of 16 osteosarcomas, 3 out of 7 ovarian cancers, 3 out of 3pancreatic cancers, 21 out of 23 prostate cancers, 12 out of 12 SCLCs,11 out of 26 soft tissue tumors and 7 out of 9 testicular tumors, ascompared with corresponding normal tissues (Table 1).

TABLE 1 Ratio of cases observed up-regulation of UBE2T in canceroustissues as compared with normal corresponding tissues. Cancer/TumorRatio Bladder cancer 24/24 Breast cancer 44/50 Cervical cancer 14/15Cholangiocellular carcinoma 12/12 CML  9/16 Colorectal cancer 9/9Esophageal cancer 31/47 Gastric cancer 5/8 Diffuse-type gastric cancer2/2 NSCLC 23/27 Lymphoma 3/3 Osteosarcoma  9/16 Ovarian cancer 3/7Pancreatic cancer 3/3 Prostate cancer 21/23 SCLC 12/12 Soft tissue tumor11/26 Testicular tumor 7/9

Prediction of HLA-A24 Binding Peptides Derived from UBE2T

Table 2a and 2b show the HLA-A24 binding 9mer and 10mer peptides ofUBE2T in the order of high binding affinity. A total of 27 peptides withpotential HLA-A24 binding ability were selected and examined todetermine the epitope peptides.

TABLE 2a HLA-A24 binding 9 mer peptides derived from UBE2T SEQ ID StartNO Position amino acid sequence Kd (nM) 1 60 RYPFEPPQI 25 2 45 PYEKGVFKL752 3 124 LMADISSEF 1463 4 133 KYNKPAFLK 2283 5 55 VIIPERYPF 3082 6 138AFLKNARQW 3317 7 6 RLKRELHML 3711 8 71 LTPIYHPNI 4077 9 131 EFKYNKPAF4692 10 62 PFEPPQIRF 7647 11 43 NTPYEKGVF 9498 12 106 TVLTSIQLL 9831SEQ ID Start NO Position amino acid sequence Binding score 13 3RASRLKREL 10.56 14 84 RICLDVLKL 8.8 15 105 ATVLTSIQL 6 16 74 IYHPNIDSA 6

TABLE 2b HLA-A24 binding 10 mer peptides derived from UBE2T SEQ ID StartNO Position amino acid sequence Kd (nM) 17 130 SEFKYNKPAF 776 18 123PLMADISSEF 5392 19 131 EFKYNKPAFL 7050 SEQ ID Start NO Positionamino acid sequence Binding score 20 133 KYNKPAFLKN 19.8 21 99RPSLNIATVL 11.2 22 154 KQKADEEEML 8 23 105 ATVLTSIQLL 7.2 24 115MSEPNPDDPL 7.2 25 177 STQKRKASQL 6 26 30 QMDDLRAQIL 5.76 27 44TPYFKGVFKL 5.28

Start position indicates the number of amino acid residue from theN-terminus of UBE2T.

Binding score and dissociation constant [Kd (nM)] are derived from“BIMAS” and “NetMHC3.2”, respectively.

Prediction of HLA-A02 Binding Peptides Derived from UBE2T

Table 3a and 3b show the HLA-A02 binding 9mer and 10mer peptides ofUBE2T in the order of high binding affinity. A total of 36 peptides withpotential HLA-A02 binding ability were selected and examined todetermine the epitope peptides.

TABLE 3a HLA-A02 binding 9 mer peptides derived from UBE2T SEQ ID StartNO Position amino acid sequence Kd (nM) 28 161 EMLDNLPEA 76 29 107VLTSIQLLM 385 30 30 QMDDLRAQI 473 31 103 NIATVLTSI 668 32 106 TVLTSIQLL1048 33 124 LMADISSEF 1595 34 6 RLKRELIIML 1653 35 101 SLNIATVLT 3347 3649 GVFKLEVII 5114 37 70 FLTPIYHPN 5950 38 13 MLATEPPPG 6039 39 84RICLDVLKL 6284 40 66 PQIRFLTPI 6587 41 132 FKYNKPAFL 6642 42 96GAWRPSLNI 8188 43 81 SAGRICLDV 8938 44 14 LATEPPPGI 9511 45 105ATVLTSIQL 9872 SEQ ID Start NO Position amino acid sequenceBinding score 46 139 FLKNARQWT 6.599

TABLE 3b HLA-A02 binding 10 mer peptides derived from UBE2T SEQ ID StartNO Position amino acid sequence Kd (nM) 47 13 MLATEPPPGI 43 48 70FLTPIYHPNI 49 49 6 RLKRELHMLA 1415 50 165 NLPEAGDSRV 1902 51 106TVLTSIQLLM 2648 52 102 LNIATVLTSI 3011 53 30 QMDDLRAQIL 4035 54 17HMLATEPPPG 4153 55 101 SLNIATVLTS 4622 56 29 DQMDDLRAQI 5029 57 105ATVLTSIQLL 5128 58 38 ILGGANTPYE 6464 59 107 VLTSIQLLMS 7911 60 161EMLDNLPEAG 9002 61 113 LLMSEPNPDD 9132 62 104 IATVLTSIQL 9157 SEQ IDStart NO Position amino acid sequence Binding score 63 44 TPYEKGVFKL24.406Start position indicates the number of amino acid residue from theN-terminus of UBE2T.

Binding score and dissociation constant [Kd (nM)] are derived from“BIMAS” and “NetMHC3.2”, respectively.

CTL Induction with the Predicted Peptides from UBE2T Restricted withHLA-A *2402

CTLs for those peptides derived from UBE2T were generated according tothe protocols as described in “Materials and Methods”. Peptide-specificCTL activity was detected by IFN-gamma ELISPOT assay (FIG. 1). It showedthat the well number #8 stimulated with UBE2T-A24-9-60 (SEQ ID NO: 1)(a), #1 stimulated with UBE2T-A24-9-45 (SEQ ID NO: 2) (b), #6 stimulatedwith UBE2T-A24-9-133 (SEQ ID NO: 4) (c), #6 stimulated withUBE2T-A24-9-138 (SEQ ID NO: 6) (d), #4 stimulated with UBE2T-A24-9-43(SEQ ID NO: 11) (e), #2 stimulated with UBE2T-A24-9-106 (SEQ ID NO: 12)(f), #6 stimulated with UBE2T-A24-9-3 (SEQ ID NO: 13) (g), #3 stimulatedwith UBE2T-A24-9-105 (SEQ ID NO: 15) (h), #2 stimulated withUBE2T-A24-10-130 (SEQ ID NO: 17) (i), #1 stimulated withUBE2T-A24-10-131 (SEQ ID NO: 19) (j), #3 stimulated withUBE2T-A24-10-133 (SEQ ID NO: 20) (k), #6 stimulated with UBE2T-A24-10-99(SEQ ID NO: 21) (1), #7 stimulated with UBE2T-A24-10-154 (SEQ ID NO: 22)(m), #8 stimulated with UBE2T-A24-10-105 (SEQ ID NO: 23) (n), #1stimulated with UBE2T-A24-10-115 (SEQ ID NO: 24) (o), #4 stimulated withUBE2T-A24-10-177 (SEQ ID NO: 25) (p) and #7 stimulated withUBE2T-A24-10-44 (SEQ ID NO: 27) (q) demonstrated potent IFN-gammaproduction as compared to the control wells. On the other hand, nospecific CTL activity was detected by stimulation with other peptidesshown in Table 2a and 2b, despite those peptides had possible bindingactivity with HLA-A*2402. As a typical case of negative data, it was notshown specific IFN-gamma production from the CTL stimulated withUBE2T-A24-9-124 (SEQ ID NO: 3) (r). As a result, it indicated that 17peptides derived from UBE2T were selected as the peptides that couldinduce potent CTLs.

CTL induction with the predicted peptides from UBE2T restricted withHLA-A*0201

CTLs for those peptides derived from UBE2T were generated according tothe protocols as described in “Materials and Methods”. Peptide-specificCTL activity was detected by IFN-gamma ELISPOT assay (FIG. 2). It showedthat the well number #4 stimulated with UBE2T-A02-9-107 (SEQ ID NO: 29)(a), #5 stimulated with UBE2T-A02-9-30 (SEQ ID NO: 30) (b), #7stimulated with UBE2T-A02-9-106 (SEQ ID NO: 32) (c), #5 stimulated withUBE2T-A02-9-49 (SEQ ID NO: 36) (d), #3 stimulated with UBE2T-A02-9-13(SEQ ID NO: 38) (e), #4 stimulated with UBE2T-A02-9-132 (SEQ ID NO: 41)(f), #6 stimulated with UBE2T-A02-10-70 (SEQ ID NO: 48) (g), #7stimulated with UBE2T-A02-10-6 (SEQ ID NO: 49) (h), #8 stimulated withUBE2T-A02-10-106 (SEQ ID NO: 51) (i), #2 stimulated withUBE2T-A02-10-102 (SEQ ID NO: 52) (j), #1 stimulated with UBE2T-A02-10-30(SEQ ID NO: 53) (k), #8 stimulated with UBE2T-A02-10-101 (SEQ ID NO: 55)(1), #5 stimulated with UBE2T-A02-10-29 (SEQ ID NO: 56) (m) and #3stimulated with UBE2T-A02-10-38 (SEQ ID NO: 58) (n) demonstrated potentIFN-gamma production as compared to the control wells. On the otherhand, no specific CTL activity was detected by stimulation with otherpeptides shown in Table 3a and 3b, despite those peptides had possiblebinding activity with HLA-A*0201. As a typical case of negative data, itwas not shown specific IFN-gamma production from the CTL stimulated withUBE2T-A02-9-161 (SEQ ID NO: 28) (o). As a result, it indicated that 14peptides derived from UBE2T were selected as the peptides that couldinduce potent CTLs.

Establishment of CTL Line and Clone Against UBE2T Derived Peptide

The cells in the well number #8 stimulated with UBE2T-A24-9-60 (SEQ IDNO: 1) (a), #1 stimulated with UBE2T-A24-9-45 (SEQ ID NO: 2) (b), #6stimulated with UBE2T-A24-9-3 (SEQ ID NO: 13) (c) and #7 stimulated withUBE2T-A24-10-44 (SEQ ID NO: 27) , which showed peptide-specific CTLactivity in IFN-gamma ELISPOT assay, were expanded and established theCTL lines. CTL activities of these CTL lines were measured by IFN-gammaELISA (FIG. 3). It demonstrated that the CTL lines showed potentIFN-gamma production against target cells pulsed with the correspondingpeptide as compared to target cells without peptide pulse. Furthermore,the CTL clones were established by limiting dilution from the CTL linesas described in “Materials and Methods”, and IFN-gamma productions fromthe CTL clones against TISI cells pulsed with corresponding peptide weremeasured by IFN-gamma ELISA. Potent IFN-gamma productions were observedfrom the CTL clones stimulated with UBE2T-A24-9-60 (SEQ ID NO: 1) (a),UBE2T-A24-9-45 (SEQ ID NO: 2) (b) and UBE2T-A24-9-3 (SEQ ID NO: 13) (c)(FIG. 4).

The cells in the well number #4 stimulated with UBE2T-A02-9-107 (SEQ IDNO: 29) (a), #3 stimulated with UBE2T-A02-9-13 (SEQ ID NO: 38) (b), #6stimulated with UBE2T-A02-10-70 (SEQ ID NO: 48) (c), #2 stimulated withUBE2T-A02-10-102 (SEQ ID NO: 52) (d) and #8 stimulated withUBE2T-A02-10-101 (SEQ ID NO: 55) (e), which showed peptide-specific CTLactivity in IFN-gamma ELISPOT assay, were expanded and established theCTL lines. CTL activities of these CTL lines were measured by IFN-gammaELISA (FIG. 5). It demonstrated that the CTL lines showed potentIFN-gamma production against target cells pulsed with the correspondingpeptide as compared to target cells without peptide pulse. Furthermore,the CTL clones were established by limiting dilution from the CTL linesas described in “Materials and Methods”, and IFN-gamma productions fromthe CTL clones against T2 cells pulsed with corresponding peptide weremeasured by IFN-gamma ELISA. Potent IFN-gamma productions were observedfrom the CTL clones stimulated with UBE2T-A02-9-107 (SEQ ID NO: 29) (a),UBE2T-A02-9-13 (SEQ ID NO: 38) (b), UBE2T-A02-10-70 (SEQ ID NO: 48) (c),UBE2T-A02-10-102 (SEQ ID NO: 52) (d) and UBE2T-A02-10-101 (SEQ ID NO:55) (e) (FIG. 6).

Specific CTL Activity Against Target Cells Expressing UBE2T andHLA-A*2402 or HLA-A*0201

The established CTL clone against UBE2T-A24-9-60 (SEQ ID NO: 1) peptidewas examined for the ability to recognize target cells that expressUBE2T and HLA-A*2402 molecule. COS7 cells transfected with both the fulllength of UBE2T and HLA-A*2402 gene (a specific model for the targetcells that express UBE2T and HLA-A*2402 gene) were prepared as astimulator cells, and COS7 cells transfected with either full length ofUBE2T or HLA-A*2402 were used as the controls. In FIG. 7, the CTL clonestimulated with UBE2T-A24-9-60 (SEQ ID NO: 1) showed potent CTL activityagainst COS7 cells expressing both UBE2T and HLA- A*2402. On the otherhand, no significant specific CTL activity was detected against thecontrols. Thus, these data clearly demonstrate that UBE2T-A24-9-60 (SEQID NO: 1) peptide is endogenously processed and expressed on the targetcells with HLA-A*2402 molecule and is recognized by the CTLs. Theseresults indicate that this peptide derived from UBE2T may be availableto apply the cancer vaccines for patients with UBE2T expressing tumors.

The established CTL line against UBE2T-A02-10-70 (SEQ ID NO: 48) peptidewas examined for the ability to recognize target cells that expressUBE2T and HLA-A*0201 molecule. COS7 cells transfected with both the fulllength of UBE2T and HLA-A*0201 gene (a specific model for the targetcells that express UBE2T and HLA-A*0201 gene) were prepared as astimulator cells, and COS7 cells transfected with either full length ofUBE2T or HLA-A*0201 were used as the controls. In FIG. 8, the CTL linestimulated with UBE2T-A02-10-70 (SEQ ID NO: 48) showed potent CTLactivity against COS7 cells expressing both UBE2T and HLA-A*0201. On theother hand, no significant specific CTL activity was detected againstthe controls. Thus, these data clearly demonstrate that UBE2T-A02-10-70(SEQ ID NO: 48) peptide is endogenously processed and expressed on thetarget cells with HLA-A*0201 molecule and is recognized by the CTLs.These results indicate that this peptide derived from UBE2T may beavailable to apply the cancer vaccines for patients with UBE2Texpressing tumors.

Homology Analysis of Antigen Peptides

The CTLs stimulated with UBE2T-A24-9-60 (SEQ ID NO: 1), UBE2T-A24-9-45(SEQ ID NO: 2), UBE2T-A24-9-133 (SEQ ID NO: 4), UBE2T-A24-9-138 (SEQ IDNO: 6), UBE2T-A24-9-43 (SEQ ID NO: 11), UBE2T-A24-9-106 (SEQ ID NO: 12),UBE2T-A24-9-3 (SEQ ID NO: 13), UBE2T-A24-9-105 (SEQ ID NO: 15),UBE2T-A24-10-130 (SEQ ID NO: 17), UBE2T-A24-10-131 (SEQ ID NO: 19),UBE2T-A24-10-133 (SEQ ID NO: 20), UBE2T-A24-10-99 (SEQ ID NO: 21),UBE2T-A24-10-154 (SEQ ID NO: 22), UBE2T-A24-10-105 (SEQ ID NO: 23),UBE2T-A24-10-115 (SEQ ID NO: 24), UBE2T-A24-10-177 (SEQ ID NO: 25),UBE2T-A24-10-44 (SEQ ID NO: 27), UBE2T-A02-9-107 (SEQ ID NO: 29),UBE2T-A02-9-30 (SEQ ID NO: 30), UBE2T-A02-9-106 (SEQ ID NO: 32),UBE2T-A02-9-49 (SEQ ID NO: 36), UBE2T-A02-9-13 (SEQ ID NO: 38),UBE2T-A02-9-132 (SEQ ID NO: 41), UBE2T-A02-10-70 (SEQ ID NO: 48),UBE2T-A02-10-6 (SEQ TD NO: 49), UBE2T-A02-10-106 (SEQ TD NO: 51),UBE2T-A02-10-102 (SEQ ID NO: 52), UBE2T-A02-10-30 (SEQ ID NO: 53),UBE2T-A02-10-101 (SEQ ID NO: 55), UBE2T-A02-10-29 (SEQ ID NO: 56) andUBE2T-A02-10-38 (SEQ ID NO: 58) showed significant and specific CTLactivity. This result may be due to the fact that the sequences ofUBE2T-A24-9-60 (SEQ ID NO: 1), UBE2T-A24-9-45 (SEQ ID NO: 2),UBE2T-A24-9-133 (SEQ ID NO: 4), UBE2T-A24-9-138 (SEQ ID NO: 6),UBE2T-A24-9-43 (SEQ ID NO: 11), UBE2T-A24-9-106 (SEQ ID NO: 12),UBE2T-A24-9-3 (SEQ ID NO: 13), UBE2T-A24-9-105 (SEQ ID NO: 15),UBE2T-A24-10-130 (SEQ ID NO: 17), UBE2T-A24-10-131 (SEQ ID NO: 19),UBE2T-A24-10-133 (SEQ ID NO: 20), UBE2T-A24-10-99 (SEQ ID NO: 21),UBE2T-A24-10-154 (SEQ ID NO: 22), UBE2T-A24-10-105 (SEQ ID NO: 23),UBE2T-A24-10-115 (SEQ ID NO: 24), UBE2T-A24-10-177 (SEQ ID NO: 25),UBE2T-A24-10-44 (SEQ ID NO: 27), UBE2T-A02-9-107 (SEQ ID NO: 29),UBE2T-A02-9-30 (SEQ ID NO: 30), UBE2T-A02-9-106 (SEQ ID NO: 32),UBE2T-A02-9-49 (SEQ ID NO: 36), UBE2T-A02-9-13 (SEQ ID NO: 38),UBE2T-A02-9-132 (SEQ ID NO: 41), UBE2T-A02-10-70 (SEQ ID NO: 48),UBE2T-A02-10-6 (SEQ ID NO: 49), UBE2T-A02-10-106 (SEQ ID NO: 51),UBE2T-A02-10-102 (SEQ ID NO: 52), UBE2T-A02-10-30 (SEQ ID NO: 53),UBE2T-A02-10-101 (SEQ ID NO: 55), UBE2T-A02-10-29 (SEQ ID NO: 56) andUBE2T-A02-10-38 (SEQ ID NO: 58) are homologous to peptide derived fromother molecules that are known to sensitize the human immune system. Toexclude this possibility, homology analyses were performed for thesepeptide sequences using as queries the BLAST algorithm(http://www.ncbi.nlm.nih.gov/blast/blast.cgi), which revealed nosequence with significant homology. The results of homology analysesindicate that the sequence of UBE2T-A24-9-60 (SEQ ID NO: 1),UBE2T-A24-9-45 (SEQ ID NO: 2), UBE2T-A24-9-133 (SEQ ID NO: 4),UBE2T-A24-9-138 (SEQ ID NO: 6), UBE2T-A24-9-43 (SEQ ID NO: 11),UBE2T-A24-9-106 (SEQ ID NO: 12), UBE2T-A24-9-3 (SEQ ID NO: 13),UBE2T-A24-9-105 (SEQ ID NO: 15), UBE2T-A24-10-130 (SEQ ID NO: 17),UBE2T-A24-10-131 (SEQ ID NO: 19), UBE2T-A24-10-133 (SEQ ID NO: 20),UBE2T-A24-10-99 (SEQ ID NO: 21), UBE2T-A24-10-154 (SEQ ID NO: 22),UBE2T-A24-10-105 (SEQ TD NO: 23), UBE2T-A24-10-115 (SEQ ID NO: 24),UBE2T-A24-10-177 (SEQ ID NO: 25), UBE2T-A24-10-44 (SEQ ID NO: 27),UBE2T-A02-9-107 (SEQ ID NO: 29), UBE2T-A02-9-30 (SEQ ID NO: 30),UBE2T-A02-9-106 (SEQ ID NO: 32), UBE2T-A02-9-49 (SEQ ID NO: 36),UBE2T-A02-9-13 (SEQ ID NO: 38), UBE2T-A02-9-132 (SEQ ID NO: 41),UBE2T-A02-10-70 (SEQ ID NO: 48), UBE2T-A02-10-6 (SEQ ID NO: 49),UBE2T-A02-10-106 (SEQ ID NO: 51), UBE2T-A02-10-102 (SEQ TD NO: 52),UBE2T-A02-10-30 (SEQ TD NO: 53), UBE2T-A02-10-101 (SEQ ID NO: 55),UBE2T-A02-10-29 (SEQ ID NO: 56) and UBE2T-A02-10-38 (SEQ ID NO: 58) areunique and thus, there is little possibility, to our best knowledge,that these molecules raise unintended immunologic response to someunrelated molecule. In conclusion, we identified novel HLA-A*2402 orHLA-A*0201 epitope peptides derived from UBE2T. Furthermore, it wasdemonstrated that epitope peptides of UBE2T may be applicable for cancerimmunotherapy.

INDUSTRIAL APPLICABILITY

The present invention provides new epitope peptides derived from UBE2Tthat may induce potent and specific anti-tumor immune responses and haveapplicability to a wide variety of cancer types. Such peptides can finduse as peptide vaccines against diseases associated with UBE2T, e.g.,cancer, more particularly, bladder cancer, breast cancer, cervicalcancer, cholangiocellular carcinoma, CML, colorectal cancer, esophagealcancer, gastric cancer, diffuse-type gastric cancer, NSCLC, lymphoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, SCLC,soft tissue tumor and testicular tumor.

While the present invention is herein described in detail and withreference to specific embodiments thereof, it is to be understood thatthe foregoing description is exemplary and explanatory in nature and isintended to illustrate the present invention and its preferredembodiments. Through routine experimentation, one skilled in the artwill readily recognize that various changes and modifications can bemade therein without departing from the spirit and scope of the presentinvention, the metes and bounds of which are defined by the appendedclaims.

1. An isolated peptide having cytotoxic T lymphocyte (CTL) inducibility,wherein the peptide comprises an amino acid sequence (a) or (b) bellow:(a) an amino acid sequence selected from the group consisting of SEQ IDNOs: 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25, 27, 29,30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58; (b) an amino acidsequence in which 1, 2, or several amino acid(s) are substituted,deleted, inserted and/or added in the amino acid sequence selected fromthe group consisting of SEQ ID NOS: 2, 4, 6, 11, 12, 13, 15, 17, 19, 20,21, 22, 23, 24, 25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55,56 and
 58. 2. The isolated peptide of claim 1, wherein the peptide isthe following oligopeptide (i) or (ii): (i) a peptide that has one orboth of the following characteristics: (a) the second amino acid fromthe N-terminus of the amino acid sequence of SEQ ID NO: 2, 4, 6, 11, 12,13, 15, 17, 19, 20, 21, 22, 23, 24, 25 or 27 is substituted withphenylalanine, tyrosine, methionine or tryptophan; and (b) theC-terminal amino acid of the amino acid sequence of SEQ ID NO: 2, 4, 6,11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25 or 27 is substituted withphenylalanine, leucine, isoleucine, tryptophan or methionine; (ii) apeptide that has one or both of the following characteristics: (a) thesecond amino acid from the N-terminus of the amino acid sequence of SEQID NO: 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 or 58 issubstituted with leucine or methionine; and (b) the C-terminal aminoacid of the amino acid sequence of SEQ ID NO: 29, 30, 32, 36, 38, 41,48, 49, 51, 52, 53, 55, 56 or 58 is substituted with valine or leucine.3. The isolated peptide of claim 1, wherein the peptide is a nonapeptideor a decapeptide.
 4. The isolated peptide of claim 1, wherein thepeptide consists of the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22,23, 24, 25, 27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and58.
 5. An isolated polynucleotide encoding the peptide of claim
 1. 6. Acomposition for inducing a CTL, wherein the composition comprises atleast one active ingredient selected from the group consisting of: (a)the peptide of claim 1; (b) the polynucleotide encoding the peptide of(a); (c) an antigen-presenting cell (APC) that presents the peptide ofclaim 1 on its surface; and (d) an exosome that presents the peptide ofclaim 1 on its surface.
 7. A pharmaceutical composition for thetreatment of cancer wherein the composition comprises at least oneactive ingredient selected from the group consisting of: (a) the peptideof claim 1; (b) the polynucleotide encoding the peptide of (a); (c) anAPC that presents the peptide of claim 1 on its surface; (d) an exosomethat presents the peptide of claim 1 on its surface; and (e) a CTL thatcan recognize a cell presenting the peptide of claim
 1. 8. Thepharmaceutical composition of claim 7, wherein the pharmaceuticalcomposition is formulated for the administration to a subject whose HLAantigen is HLA A241 or HLA A2.
 9. A method for inducing an APC with CTLinducibility, wherein the method comprises the step selected from thegroup consisting of: (a) contacting an APC with the peptide of claim 1,and (b) introducing a polynucleotide encoding the peptide of claim 1into an APC.
 10. A method for inducing a CTL, wherein the methodcomprises a step selected from the group consisting of: (a) co-culturinga CD8 positive T cell with an APC that presents on its surface a complexof an HLA antigen and the peptide of claim 1; (b) co-culturing a CD8positive T cell with an exosome that presents on its surface a complexof an HLA antigen and the peptide of claim 1; and (c) introducing into aCD8 positive T cell a polynucleotide encoding both of T cell receptor(TCR) subunits or polynucleotides encoding each of TCR subunits, whereinthe TCR formed by said subunits can bind to a complex of the peptide ofclaim 1 and an HLA antigen on a cell surface.
 11. An isolated APC thatpresents on its surface a complex of an HLA antigen and the peptide ofclaim
 1. 12. An isolated APC which is induced by the method comprising;a step selected from the group consisting of: (a) contacting an APC withthe peptide of claim 1 in vitro, ex vivo or in vivo, and (b) introducinga polynucleotide encoding the peptide of claim 1 into an APC.
 13. Anisolated CTL that targets the peptide of claim
 1. 14. An isolated CTLwhich is induced by the method comprising a step selected from the groupconsisting of: (a) co-culturing a CD8 positive T cell with an APC thatpresents on its surface a complex of an HLA antigen and the peptide ofclaim 1; (b) co-culturing a CD8 positive T cell with an exosome thatpresents on its surface a complex of an HLA antigen and the peptide ofclaim 1; and (c) introducing into a CD8 positive T cell a polynucleotideencoding both of T cell receptor (TCR) subunits or polynucleotidesencoding each of TCR subunits, wherein the TCR formed by said subunitscan bind to a complex of the peptide of claim 1 and an HLA antigen on acell surface.
 15. A method of inducing an immune response against cancerin a subject, wherein the method comprises the step of administering tothe subject a composition comprising the peptide of claim 1, or apolynucleotide encoding the peptide. 16-19 (canceled)
 20. A method ofscreening for a peptide having an ability to induce a CTL, that hasspecific cytotoxic activity against a cell that presents a fragmentderived from UBE2T, wherein the method comprises the steps of: (i)providing a candidate sequence consisting of an amino acid sequencemodified by substituting, deleting, inserting and/or adding one, two orseveral amino acid residues to an original amino acid sequence, whereinthe original amino acid sequence is selected from the group consistingof SEQ ID NOS: 2, 4, 6, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 25.27, 29, 30, 32, 36, 38, 41, 48, 49, 51, 52, 53, 55, 56 and 58; (ii)selecting a candidate sequence that does not have substantialsignificant homology with the peptides derived from any known human geneproducts other than UBE2T; (iii) contacting a peptide consisting of thecandidate sequence selected in step (ii) with an antigen presentingcell; (iv) contacting the antigen presenting cell of step (iii) with aCD8 positive T cell; and (v) identifying the peptide of which CTLinducibility is same to or higher than a peptide consisting of theoriginal amino acid sequence.